Associations of plasma fibrinogen assays, C-reactive protein and interleukin-6 with previous myocardial infarction

Summary. Background: The association of plasma fibrinogen with myocardial infarction (MI) may (like that of C‐reactive protein, CRP) be a marker of subclinical inflammation, mediated by cytokines such as interleukin‐6 (IL‐6). There are well‐recognized discrepancies between commonly performed fibrinogen assays. Increased ratio of clottable fibrinogen to intact fibrinogen (measured by a recently developed immunoassay) has been proposed as a measure of hyperfunctional fibrinogen, and is elevated in acute MI. Objective: To compare the associations of intact fibrinogen and four routine fibrinogen assays (two von Clauss assays; one prothrombin‐time derived; and one immunonephelometric) in a case–control study of previous MI. Patients/methods: Cases (n = 399) were recruited 3–9 months after their event; 413 controls were age‐ and sex‐ matched from the case–control study local population. Intact fibrinogen was measured in 50% of subjects. Results: All routine fibrinogen assays showed high intercorrelations (r = 0.82–0.93) and significant (P < 0.0001) increased mean levels in cases vs. controls. These four routine assays correlated only moderately with intact fibrinogen (r = 0.45–0.62), while intact fibrinogen showed only a small, nonsignificant increase in cases vs. controls. Consequently, the ratio of each of the four routine assays to the intact fibrinogen assay was significantly higher (P < 0.0003) in cases vs. controls. Each fibrinogen assay correlated with plasma levels of CRP and IL‐6 (which were also elevated in cases vs. controls). Each routine fibrinogen assay remained significantly elevated in cases vs. controls after further adjustment for C‐reactive protein and interleukin‐6. Conclusions: These data provide evidence for acquired, increased hyperfunctional plasma fibrinogen in MI survivors, which is not associated with markers of inflammatory reactions. The causes and significance of these results remain to be established in prospective studies.     

Involvement of the β3 E749ATSTFTN756 region in stabilizing integrin αIIbβ3-ligand interaction

Summary. Platelet integrin α_IIbβ₃ must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface‐bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the β‐subunit plays a central role. We introduced peptides homologous to the E⁷⁴⁹ATSTFTN⁷⁵⁶ domain (E–N peptide) and the T⁷⁵⁵NITYRGT⁷⁶² domain (T–T peptide) of β₃ in streptolysin O‐permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC‐1 after stimulation with thrombin. E–N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E–N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E–N peptide did not disturb the binding of PAC‐1, which is known to reflect activation of the integrin. E–N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on α_IIbβ₃. T–T peptide did not affect these processes. In a model for outside‐in integrin activation, E–N peptide disrupted the binding of CHO cells expressing α_IIbβ₃ to surface‐bound ligand. Again, T–T peptide had no effect. We conclude that the E⁷⁴⁹ATSTFTN⁷⁵⁶ region of the β₃‐tail stabilizes the binding of soluble and surface‐bound ligand to integrin α_IIbβ₃ via a mechanism that involves the phosphorylation of FAK.     

Longitudinal assessment of fibrinogen in relation to subclinical cardiovascular disease: the CARDIA study

Summary. Objective: To examine the strength of the associations of fibrinogen with subclinical atherosclerosis in healthy persons. Methods: A population‐based, prospective, observational study of black and white men and women (Coronary Artery Risk Development in Young Adults [CARDIA]). Fibrinogen levels were measured at year 7 (ages 25–37, n = 2969), and again at year 20 (ages 38–50, n = 2832). Measures of subclinical atherosclerosis (coronary artery calcification [CAC] and carotid intimal‐medial thickness [CIMT]) were recorded at year 20. Results: Over the 13‐year study interval (1992–1993 to 2005–2006), fibrinogen rose from a mean of 3.32 to 4.05 g L^?1. After adjusting for age, gender and race, fibrinogen was positively associated with greater incidence of CAC and increased CIMT cross‐sectionally as well as after 13 years of follow‐up (all P‐trend < 0.001). After further adjustment for field center, BMI, smoking, education, systolic blood pressure, diabetes, antihypertensive medication use, total and HDL cholesterol, and CRP, significant positive relationships between fibrinogen and incidence of CAC remained for the total cohort longitudinally (P‐trend = 0.037), but not cross‐sectionally (P‐trend = 0.147). Conclusion: This 13‐year study demonstrates that higher levels of fibrinogen during young adulthood are positively associated with incidence of CAC and increased CIMT in middle‐age, but the strength of the association declines with increasing age.     

Identification of Hic‐5 as a novel regulatory factor for integrin αIIbβ3 activation and platelet aggregation in mice

Summary. Background: Integrin αIIbβ3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide‐inducible clone‐5 (Hic‐5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic strand. Objectives: Hic‐5 function in αIIbβ3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic‐5^?/? mice were analyzed. Methods and Results: Hic‐5^?/? mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin‐induced aggregation in Hic‐5^?/? platelets. Mechanistically, Hic‐5^?/? platelets showed limited activation of αIIbβ3 upon thrombin treatment. Morphological alteration in Hic‐5^?/? platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co‐immunoprecipitating with the activated αIIbβ3 was smaller in Hic‐5^?/? platelets than in wild‐type platelets. Conclusion: We identified Hic‐5 as a novel and specific regulatory factor for thrombin‐induced αIIbβ3 activation and subsequent platelet aggregation in mice.     

Fibrinogen counteracts the antiadhesive effect of fibrin‐bound plasminogen by preventing its activation by adherent U937 monocytic cells

Summary. Background: Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface‐mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non‐adhesive, while the conversion of surface‐bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment. Objectives: Since fibrin clots in circulation are exposed to both fibrinogen and plasminogen, their combined effect on adhesion of monocytic cells was examined. Methods: Fibrin gels were coated with plasminogen and its activation by adherent U937 monocytic cells in the presence of increasing concentrations of fibrinogen was examined by either measuring ¹²⁵I‐labeled fibrin degradation products or plasmin amidolytic activity. Results: Unexpectedly, the antiadhesive effects of two fibrin binding proteins were not additive; in fact, in the presence of fibrinogen, the effect of plasminogen was strongly reduced. An investigation of the underlying mechanism revealed that fibrinogen prevented activation of fibrin‐bound plasminogen by cells. Confocal microscopy showed that fibrinogen accumulates in a thin superficial layer of a clot, where it exerts its blocking effect on activation of plasminogen. Conclusion: The results point to a complex interplay between the fibrinogen‐ and plasminogen‐dependent antiadhesive systems, which may contribute to the mechanisms that control the adhesiveness of a fibrin shell on the surface of hemostatic thrombi.     

Overexpression of the partially activated αIIbβ3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet–like extensions in Chinese hamster ovary cells

Summary. Background: We have recently reported a novel mutation in the β₃ subunit of the platelet fibrinogen receptor (α_IIbβ₃D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen‐dependent, microtubule‐driven proplatelet‐like cell extensions. Results: Here we demonstrate that the partially activated α_IIbβ₃D723H or α_IIbβ₃D723A salt bridge mutants, but not fully activated α_IIbβ₃ mutants, cause this phenotype. Time‐lapse videomicroscopy clearly differentiated these stable microtubule‐driven and nocodazole‐sensitive extensions from common dynamic actin‐driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of α_IIbβ₃, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34⁺‐derived megakaryocyte proplatelets. Mutant α_IIbβ₃D723H signaling was independent of Src, protein kinase C or phosphoinositide 3‐kinase, but correlated with decreased RhoA activity as compared with wild‐type α_IIbβ₃ signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO α_IIbβ₃D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO α_IIbβ₃D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin‐dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule‐driven formation of cytoplasmic extensions.     

Integrin α2β1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cγ2 in platelets: role of Src kinase and Rac GTPase

Summary. Background: Platelet adhesion promoted by integrin  α₂β₁ induces integrin  α_IIbβ₃ activation through the phospholipase C (PLC)‐dependent stimulation of the small GTPase Rap1b. Objective: To analyze the mechanism of PLC activation downstream of α₂β₁ that is required for regulation of Rap1b and α_IIbβ₃. Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through α₂β₁. Tyrosine phosphorylation of PLCγ2, PLC activation, accumulation of GTP‐bound Rap1b and fibrinogen binding were measured and compared. Results: Integrin  α₂β₁ recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCγ2, and was suppressed in platelets from PLCγ2‐knockout mice. Moreover, PLCγ2^?/? platelets were unable to accumulate active Rap1b and to activate α_IIbβ₃ upon adhesion through α₂β₁. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCγ2 in adherent platelets, but did not affect its activation, and both Rap1b and α_IIbβ₃ stimulation occurred normally. Importantly, α_IIbβ₃‐induced phosphorylation and activation of PLCγ2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin  α₂β₁ recruitment triggered the Src kinase‐independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCγ2 phosphorylation. However, when phosphorylation of PLCγ2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCγ2 activation, GTP‐Rap1b accumulation, and α_IIbβ₃ stimulation. Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLCγ2 activation downstream of α₂β₁.     

French lyophilized plasma versus fresh frozen plasma for the initial management of trauma‐induced coagulopathy: a randomized open‐label trial

Essentials

• An immediate supply of plasma in case of trauma‐induced coagulopathy is required.
• The Traucc trial compared French Lyophilised Plasma (FLyP) and Fresh Frozen Plasma (FFP).
• FLyP achieved higher fibrinogen concentrations compared with FFP.
• FLyP led to a more rapid coagulopathy improvement than FFP.

Summary

Background

Guidelines recommend beginning hemostatic resuscitation immediately in trauma patients. We aimed to investigate if French lyophilized plasma (FLyP) was more effective than fresh frozen plasma (FFP) for the initial management of trauma‐induced coagulopathy.

Methods

In an open‐label, phase 3, randomized trial (NCT02750150), we enrolled adult trauma patients requiring an emergency pack of 4 plasma units within 6 h of injury. We randomly assigned patients to receive 4‐FLyP units or 4‐FFP units. The primary endpoint was fibrinogen concentration at 45 min after randomization. Secondary outcomes included time to transfusion, changes in hemostatic parameters at different time‐points, blood product requirements and 30‐day in‐hospital mortality.

Results

Forty‐eight patients were randomized (FLyP, n = 24; FFP, n = 24). FLyP reduced the time from randomization to transfusion of first plasma unit compared with FFP (median[IQR],14[5–30] vs. 77[64–90] min). FLyP achieved a higher fibrinogen concentration 45 min after randomization compared with FFP (baseline‐adjusted mean difference, 0.29 g L^?1; 95% confidence interval [CI], 0.08–0.49) and a greater improvement in prothrombin time ratio, factor V and factor II. The between‐group differences in coagulation parameters remained significant at 6 h. FLyP reduced fibrinogen concentrate requirements. Thirty‐day in‐hospital mortality rate was 22% with FLyP and 29% with FFP.

Conclusion

FLyP led to a more rapid, pronounced and extended increase in fibrinogen concentrations and coagulopathy improvement compared with FFP in the initial management of trauma patients. FLyP represents an attractive option for trauma management, especially when facing logistical issues such as combat casualties or mass casualties related to terror attacks or disasters.

     

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

Summary. Background: β₃‐Integrins are involved in platelet aggregation via α_IIbβ₃ [glycoprotein (GP)IIb–GPIIIa], and in angiogenesis via endothelial α_Vβ₃. Cross‐reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. Objectives: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on β₃‐integrins. Methods: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC₅₀ were determined by immunodot assays, enzyme‐linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb–GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. Results: We observed specific and high‐affinity binding to an intact GPIIb–GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose‐dependent fibrinogen binding to GPIIb–GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross‐reactivity with α_Vβ₃, and also demonstrated proangiogenic effects in tube formation and CAM assays. Conclusions: These Fab fragments are the first entirely human anti‐GPIIb–GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual‐specificity anti‐β₃‐integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.     

Pharmacokinetics and safety of fibrinogen concentrate

Summary. Background: Although fibrinogen concentrate has been available for the treatment of congenital fibrinogen deficiency for years, knowledge of its pharmacokinetics comes from only two small studies. Objectives: To assess the pharmacokinetic (PK) profile, clot integrity and safety of fibrinogen concentrate (human) (FCH) in patients with afibrinogenemia. Patients and methods: A multinational, prospective, open‐label, uncontrolled study of patients with afibrinogenemia ≥ 6 years of age was conducted in the USA and Italy. Plasma was collected before and after infusion for PK analyses and evaluation by rotational thromboelastometry of maximum clot firmness (MCF) to assess clot integrity. Safety was assessed on the basis of adverse events and laboratory parameters. Results: After a single dose of 70 mg kg^?1 body weight (b.w.) FCH in 14 patients, median incremental in vivo recovery was a 1.7 mg dL^?1 increase per mg kg^?1 b.w., and median levels were 1.3 g L^?1 for fibrinogen activity and antigen 1 h after infusion. Median half‐life (t_1/2) was 77.1 h for fibrinogen activity and 88.0 h for antigen. Plasma recovery in children < 16 years old was similar to that in adults aged 16 to < 65 years, but the t_1/2 and area under the curve were decreased, with an increased steady‐state volume and clearance. MCF increased by a mean of 8.9 mm from baseline to 1 h after infusion of FCH (P < 0.0001). All four adverse events reported were mild, and none was serious or related to study drug. Conclusions: These PK findings confirm a rapid increase in plasma fibrinogen levels after infusion with FCH. Together with the clot integrity and safety data and published data on efficacy, the results support the idea that FCH substitution can restore hemostasis with a good safety profile.     

Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: implications for the mechanism of thrombosis in hyperhomocysteinemia

Summary. Elevated plasma homocysteine is associated with an increased risk of atherosclerosis and thrombosis. However, the mechanisms by which homocysteine might cause these events are not understood. We hypothesized that hyperhomocysteinemia might lead to modification of fibrinogen in vivo, thereby causing altered fibrin clot structure. New Zealand White rabbits were injected intraperitoneally (i.p.) every 12 h through an indwelling catheter with homocysteine or buffer for 8 weeks. This treatment raised the plasma homocysteine levels to about 30 µmol L^?1 compared with 13.5 µmol L^?1 in control rabbits by the end of the treatment period. The fibrinogen levels were 3.2 ± 0.6 in homocysteine‐treated and 2.5 ± 1.1 mg mL^?1 in control rabbits. The reptilase time was prolonged to 363 ± 88 for plasma from homocysteine‐treated rabbits compared with 194 ± 48 s for controls (P < 0.01). The thrombin clotting time (TCT) for the homocysteine‐treated rabbits was significantly shorter, 7.5 ± 1.7 compared with 28.6 ± 18 s for the controls (P < 0.05). The calcium dependence of the thrombin clotting time was also different in homocysteinemic and control plasmas. Clots from plasma or fibrinogen of homocysteinemic rabbits were composed of thinner fibers than control clots. The clots formed from purified fibrinogen from homocysteine‐treated rabbits were lyzed more slowly by plasmin than comparable clots from control fibrinogen. Congenital dysfibrinogenemias have been described that are associated with fibrin clots composed of thin, tightly packed fibers that are abnormally resistant to fibrinolysis, and recurrent thrombosis. Our results suggest that elevated plasma homocysteine leads to a similar acquired dysfibrinogenemia. The formation of clots that are abnormally resistant to fibrinolysis could directly contribute to the increased risk of thrombosis in hyperhomocysteinemia.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

Glycoprotein Ib–IX-mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion

Summary. The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to α_IIbβ₃, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib–IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF–GP Ib–IX and fibrinogen–α_IIbβ₃ bonds using optical tweezers. In our system, fusion of tandem repeats of FK506‐binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib–IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell‐permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma‐derived VWF or the VWF A1 domain and GP Ib–IX(FKBP)₂, and those between fibrinogen‐coated beads and α_IIbβ₃ expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib–IX(FKBP)₂ and A1 or plasma‐derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and α_IIbβ₃ was not changed by the clustering agent; however, the strength of single fibrinogen–α_IIbβ₃ bonds increased significantly after ligation of GP Ib–IX(FKBP)₂ by A1. These results demonstrate that GP Ib–IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib–IX can activate α_IIbβ₃, thereby increasing the strength of its interaction with fibrinogen.     

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary

Summary. Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL⁻¹) and adenosine diphosphate (ADP 2 and 5 μm ) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL⁻¹ ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL⁻¹. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL⁻¹ in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.     

A novel automated microchip flow‐chamber system to quantitatively evaluate thrombus formation and antithrombotic agents under blood flow conditions

Summary. Background and aims: In the present study, we describe a newly developed microchip‐based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions. Methods: Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600 s^?1, and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T₁₀ (time to 10 kPa), T_10–80 (time from 10 to 80 kPa) and OT (occlusion time; time to 80 kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively. Results: After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5–1.0 U mL^?1) potently prolonged T₁₀ at both shear rates, whereas low molecular weight heparin (1.0–2.0 IU mL^?1) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1–2 μg mL^?1) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5 U mL^?1). OS‐1 (specific GPIbα‐antagonist) prevented the complete capillary occlusion. Conclusion: The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination.     

A phase 1 ascending dose study of a subcutaneously administered factor IXa inhibitor and its active control agent

Summary. Background: The REG2 anticoagulation system consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous control agent, anivamersen. Objectives: To assess the safety, tolerability and pharmacokinetic and pharmacodynamic responses of REG2. Patients/Methods: In this phase 1a study, 36 healthy volunteers were enrolled into five cohorts and given one dose of pegnivacogin. Cohorts 1 (n = 6) and 1A (n = 4) received 0.5 mg kg^?1; cohort 2 (n = 6) received 1.0 mg kg^?1; cohort 3 (n = 6) received 3.0 mg kg^?1; and cohort 4 (n = 8) received 2.0 mg kg^?1. In cohorts 1–3, two subjects were randomized to placebo. Cohort 4 subjects were subsequently randomized to single‐dose (n = 4) or multidose (n = 4) anivamersen. Results: The mean maximum observed concentrations of pegnivacogin in cohorts 1, 1A, 2 and 3 at median time were 5.16 μg mL^?1 at 84 h, 5.19 μg mL^?1 at 72 h, 9.32 μg mL^?1 at 90 h, and 32.5 μg mL^?1 at 84 h, respectively. The maximum relative activated partial thromboplastin time and time needed to achieve this were 1.18 at 2 days, 1.16 at 2 days, 1.27 at 3 days, and 1.85 at 2 days, respectively. The calculated mean half‐life and mean residence times of pegnivacogin were 6.12 days and 9.6 days, respectively. There was rapid reversal with intravenous anivamersen, although subsequent reaccumulation of pegnivacogin was observed. Conclusions: In our first‐in‐human study, REG2 was well tolerated and provided dose‐proportional anticoagulation for several days after a single subcutaneous dose, with complete, although transient, reversal by its control agent. This study demonstrates the first application of a subcutaneously administered aptamer, and represents a potential advance in aptamer therapeutics.     

Effect of combined treatment with immunoadsorption and membrane filtration on plasma coagulation—Results of a randomized controlled crossover study

The combined use of immunoadsorption (IA) and membrane filtration (MF) may markedly enhance removal of IgM and complement component C1q, supporting its use as an element of recipient desensitization in antibody‐incompatible transplantation. However, coagulation factor removal may contribute to altered hemostasis, posing a risk of bleeding in the perioperative setting. This secondary endpoint analysis of standard coagulation assays and rotational thromboelastometry ( ROTEM® ) was performed in the context of a randomized controlled crossover study designed to assess the effect of combined IA (GAM‐146‐peptide) and MF on levels of ABO antigen‐specific IgM. Fourteen patients with autoimmune disorders were randomized to a single treatment with IA+MF followed by IA alone, or vice versa. MF was found to markedly enhance fibrinogen depletion (57% vs. 28% median decrease after IA alone, P < 0.001), whereby four patients showed post‐treatment fibrinogen concentrations below 100 mg dL^?1. In support of a critical contribution of fibrinogen depletion to impaired coagulation, extrinsically activated ROTEM^® analysis revealed a marked reduction in fibrinogen‐dependent clot formation upon IA+MF (59% median decrease in FIBTEM mean clot firmness (MCF) as compared to 24% after IA alone, P < 0.001). Moreover, the addition of MF led to a substantial prolongation of activated partial thromboplastin time, possibly due to depletion of macromolecular coagulation factors contributing to intrinsically activated coagulation. Our study demonstrates substantial effects of combined IA+MF on clot formation, which may be mainly attributable to fibrinogen depletion. We suggest that the use of combined apheresis in the setting of transplant surgery may necessitate a careful monitoring of coagulation. J. Clin. Apheresis 31:29–37, 2016. © 2015 Wiley Periodicals, Inc.     

Plasmic degradation modulates activity of fibrinogen-bound fibroblast growth factor-2

Summary. Fibroblast growth factor‐2 (FGF‐2) binds to fibrin(ogen) with high affinity, and fibrinogen potentiates FGF‐2‐stimulated proliferation of endothelial cells. Because plasmin degrades fibrin(ogen) physiologically and could liberate growth factor from fibrin deposits or alter its activity, we have now investigated the effect of plasmic degradation on the activity of fibrin(ogen)‐bound FGF‐2. Fibrinogen with bound FGF‐2 was incubated with plasmin, the products characterized by SDS–PAGE, and the proliferative activity determined by ³H‐thymidine incorporation into endothelial cells. Before plasmin exposure, proliferation was increased 3.7 ± 0.6‐fold with fibrinogen‐bound FGF‐2 compared with medium alone (P < 0.005). Plasmic degradation resulted in progressive decrease in the proliferative capacity, with the 60‐min digest showing predominantly fragment D1 and E and ³H‐thymidine uptake of only 1.2 ± 0.2‐fold, significantly less than the activity of an equal concentration of free FGF‐2 (P < 0.02). However, further degradation increased activity, and proliferation with a 90‐min digest increased to 2.6 ± 0.5‐fold, significantly greater than the 60‐min digest (P < 0.02). Plasmic degradation in the presence of 10 mm calcium chloride prevented degradation of D1 to D2 and D3, and the activity did not increase with extended degradation. Immunoprecipitation of the digests with antifibrinogen antibody showed 70 ± 8% of fibrinogen‐bound FGF‐2 in the presence of calcium but only 15 ± 4% in its absence, indicating that cleavage of D1 to D2 and D3 is critical in binding. Fragment D1 and D2, but not D3, bound to a column containing immobilized FGF‐2, indicating that a binding site is lost upon degradation to D3. The results demonstrate that plasmic degradation of fibrinogen modulates the activity and binding of FGF‐2 that involves a site near the carboxyl terminus of the γ chain.     

Aspirin and the in vitro linear relationship between thromboxane A2‐mediated platelet aggregation and platelet production of thromboxane A2

Summary. Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A₂ formation is non‐linear and TXA₂ generation must be inhibited by at least 95% to inhibit TXA₂‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA₂ generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA₂ production in response to arachidonic acid (0.03–1 mmol L^?1), collagen (0.1–30 μg mL^?1), epinephrine (0.001–100 μmol L^?1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L^?1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA₂ production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC₅₀ values that did not differ. A linear relationship existed between aggregation and TXA₂ production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R² 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA₂ generation and TXA₂‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.     

Differential response of platelets to chemokines: RANTES non-competitively inhibits stimulatory effect of SDF-1α

Summary. Background: Among the chemokines related to CXC and CC receptor groups and released from platelets, leukocytes and endothelial cells, SDF‐1, TARC and MDC have been found to be platelet agonists. Platelets do not contain SDF‐1α. In contrast, RANTES is constitutively present in platelet α‐granules and released upon platelet activation. Objectives: To study a possible role of RANTES as a modulator of SDF‐1α effect on platelets, in relation to CXCR4 and various CC receptors. Methods: CXCR‐4 (CXCL12) receptor expression and platelet activation were evaluated by flow cytometry, platelet deposition was studied by cone and plate(let) analyzer, and platelet aggregation by turbidometric aggregometry. Results: Flow cytometry studies revealed similar expression of CXCR‐4, the specific receptor of SDF‐1α on intact, inactivated, and activated platelets. Preincubation of platelets with RANTES affected neither CXCR‐4 expression, nor SDF‐1α binding to the platelet membrane. In the presence of fibrinogen, SDF‐1α activated gel‐filtered platelets. RANTES did not activate platelets, but substantially (by 70%) inhibited SDF‐1α‐induced fibrinogen binding. Similarly, RANTES abrogated the promoting effect of SDF‐1α on whole blood platelet adhesion to endothelial cell monolayer under venous flow conditions. In platelet‐rich plasma, RANTES moderately inhibited SDF‐1α‐induced platelet aggregation, while it did not affect aggregation induced by thrombin‐receptor activation peptide, adenosine diphosphate, or phorbol 12‐myristate 13‐acetate. A synergistic inhibitory effect of RANTES and prostaglandin E₁ used at subthreshold concentrations, on SDF‐1α‐induced aggregation and SDF‐1α‐induced fibrinogen binding to platelets was observed, which may suggest involvement of RANTES in a cAMP‐dependent signal transduction pathway. Conclusions: RANTES non‐competitively inhibits activation of platelets by SDF‐1α, and thus may play a regulatory role in platelet response to inflammation.