Development of fibrinogen γ-chain peptide-coated, adenosine diphosphate-encapsulated liposomes as a synthetic platelet substitute

Summary.  Background : The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen γ-chain carboxy-terminal sequence (γ400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. Objectives : To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Methods and results : Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion : H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.     

Effects of in vitro adult platelet transfusions on neonatal hemostasis

Summary. Background: Thrombocytopenia is frequent among neonates, and 20–25% of affected infants are treated with platelet transfusions. These are frequently given for mild thrombocytopenia (platelets: 50–100 × 10⁹ L^?1), largely because of the known hyporeactivity of neonatal platelets. In tests of primary hemostasis, however, neonates have shorter bleeding and closure times (CTs) than adults. This has been attributed to their higher hematocrits, higher von Willebrand factor (VWF) concentrations, and predominance of longer VWF polymers. Objective: To determine whether the ‘transfusion’ of adult (relatively hyperreactive) platelets into neonatal blood results in a hypercoagulable profile. Methods: Cord blood (CB) and adult peripheral blood (PB) were separated (with a modified buffy coat method) to generate miniaturized platelet concentrates (PCs) and thrombocytopenic blood. PB‐derived and CB‐derived PCs (n = 7 per group) were then ‘transfused’in vitro into thrombocytopenic CB and PB. The effects of autologous vs. allogeneic (developmentally mismatched) ‘transfusions’ were evaluated with whole blood aggregometry, a platelet function analyzer (PFA‐100), and thromboelastography (TEG). Results: Adult platelets aggregated significantly better than neonatal platelets in response to thrombin receptor‐activating peptide, ADP, and collagen, regardless of the blood into which they were transfused. The ‘transfusion’ of adult platelets into thrombocytopenic CB resulted in shorter CTs‐EPI (PFA‐100) and higher clot strength and firmness (TEG) than ‘transfusion’ of neonatal autologous platelets. Conclusions: In vitro‘transfusion’ of adult platelets into neonatal blood results in shorter CTs than ‘transfusion’ with neonatal platelets. Our findings should raise awareness of the differences between the neonatal and adult hemostatic system and the potential ‘developmental mismatch’ associated with platelet transfusions for neonatal hemostasis.     

人源性血小板替代物的成分测定和止血效果观察

目的研究人源性血小板替代物的主要成分和止血效果。方法采用冷冻、融化裂解和干燥技术将来源于人的过期浓缩血小板制备成血小板替代物。应用生物化学方法、流式细胞技术分析其主要成分和检测是否含有ABO血型抗原和HLA-I抗原。采用Russel蝰蛇毒时间测定(RVVT)检测其促凝血活性,并在血小板减少兔体内观测血小板替代物的止血效果。结果血小板替代物主要成分是糖蛋白(GPIb、GPⅡb和GPⅢa)和磷脂组分(磷脂酰胆碱和磷脂酰乙醇胺),不含ABO血型抗原和HLA～I抗原物质;血小板替代物RVVT为14．82s,比正常对照的12．64s延长2．18s;血小板减少兔注射血小板替代物后2、4和6小时,其出血时间比注射前明显缩短。结论人源性血小板替代物主要含糖蛋白和磷脂组分,不含ABO血型抗原和HLA-I抗原物质,其在血小板减少兔体内具有明显的短期止血活性。     

Hemostatic Function, Survival, and Membrane Glycoprotein Changes in Young versus Old Rabbit Platelets

Although in vitro studies have demonstrated functional differences between young and old platelets, in vivo differences have not been precisely established. Therefore the in vivo hemostatic function of young and old platelets and the survival time have been examined in rabbits. The hemostatic function was measured by performing serial ear bleeding times in irradiation-induced thrombocytopenic rabbits. After irradiation with 930 rad the platelet count gradually diminished reaching a nadir (~20 × 10³/μl) at 10 d. The platelets present in the circulation, 7-10 d after irradiation, were considered old platelets, and the platelets present after recovery, 11-14 d postirradiation, young platelets. The measurement of platelet size was consistent with the hypothesis that platelets become smaller with age: the mean size was 3.84 μm³ for old platelets and 5.86 μm³ for young platelets. Regression analysis of the relationship between the bleeding time and the platelet count in 18 rabbits showed a significantly different slope for rabbits with predominantly old platelets compared with rabbits with predominantly young platelets (P < 0.001). Young platelets were more effective giving much shorter bleeding times than old platelets at comparable platelet counts. Survival times of young and old platelets were measured using platelets harvested on day 8 postirradiation (old platelets) and day 12 postirradiation (young platelets) that were labeled and then reinjected into normal recipient animals. The mean platelet survival time, calculated by gamma function, of old platelets was 28.8 h; of young platelets, 87.4 h; and of normally circulating heterogeneous platelets, (normal platelets) 53.0 h. Notably, the survival of old platelets was found to be exponential, and of young platelets, linear. Analysis of the membrane glycoproteins in young, old and normal platelets indicated that there was no qualitative difference amongst the young, normal, and old platelets. The relative relationship among all the glycoprotein peaks was equal and the only changes observed were quantitative, with young platelets having significantly more membrane glycoprotein per cell than old platelets and normal platelets. Normal platelets had intermediate concentrations of each glycoprotein. These results demonstrate that young platelets are hemostatically more effective in vivo than old platelets. The data are compatible with the hypothesis that platelets age in the circulation by losing membrane fragments and then after becoming senescent, are removed from the circulation by a random process.     

Homozygous F5 deep‐intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet‐rich plasma

Summary. Background: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet‐rich plasma (PRP). Therefore, we hypothesized that FV‐deficient patients with severe bleeding manifestations may lack platelet FV. Objectives: To characterize a FV‐deficient patient with a severe bleeding diathesis. Patients/Methods: We performed FV mutation screening and functional studies in a 31‐year‐old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. Results: The proband was homozygous for a deep‐intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo‐exon with an in‐frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband’s plasma or platelets with a prothrombinase‐based assay. Moreover, no thrombin generation was observed in PRP triggered with 1–50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband’s whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. Conclusions: This is the first report of a pathogenic deep‐intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.     

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.     

Reversal of the anti‐platelet effects of aspirin and clopidogrel

Summary. Background: Guidelines recommend stopping aspirin and clopidogrel 7 to 10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets.Objectives: The purpose of the present study was to determine the rate of offset of the anti‐platelet effects of aspirin and clopidogrel after stopping treatment and the proportion of untreated donor platelets that are required to reverse their anti‐platelet effects.Methods: Cohort 1 consisted of 15 healthy subjects who received aspirin 81 mg day^?1 or clopidogrel 75 mg day^?1 for 7 days and underwent serial blood sampling until platelet function testing results normalized. Cohort 2 consisted of 36 healthy subjects who received aspirin 325 mg day^?1, clopidogrel 75 mg day^?1, aspirin 81 mg day^?1 plus clopidogrel 75 mg day^?1 or no treatment for 7 days and underwent a single blood sampling.Results: In cohort 1, arachidonic acid (AA)‐induced light transmission aggregation (LTA) returned to baseline levels in all subjects within 4 days of stopping aspirin, coinciding with the partial recovery of plasma thromboxane B₂ concentrations. ADP‐induced LTA did not return to baseline levels until 10 days after stopping clopidogrel. In cohort 2, AA‐induced LTA in patient treated with aspirin reached control levels after mixing with 30% untreated donor platelets whereas ADP‐induced LTA in patients treated with clopidogrel reached control levels only after the addition of 90% or more donor platelets.Conclusions: Platelet aggregation recovers within 4 days of stopping aspirin but clopidogrel must be stopped for 10 days to achieve a normal aggregatory response.     

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.     

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3‐kinase

Summary. Background and objective: Salvianolic acid A (SAA) is a water‐soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results: SAA dose‐dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP‐enhanced platelet P‐selectin expression and fibrinogen binding, which consequently hampered ADP‐induced platelet–leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside‐in signaling. Under an arterial shear rate of 1000 s^?1, SAA decreased platelet adhesion on collagen surfaces by ～ 40%. Western blot analysis showed that SAA, like the phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and TGX‐221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild‐type mice (35 ± 2 min without SAA and 56 ± 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr^tm1Her mutant mice (21 ± 2 min without SAA and 45 ± 4 min with SAA; P < 0.01). Conclusions: SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.     

Certificate of analysis NFKK Reference Serum X: a reprint

Objective. The Cephotest® is an activated partial thromboplastin time (APTT) test used to measure the activity of the intrinsic pathway of coagulation. To perform this test, blood is usually centrifuged to obtain plasma that is almost without erythrocytes and leucocytes and with only a minimal amount of platelets. Material and methods. In the present experiments blood was centrifuged at different speeds to produce either platelet‐poor plasma (PPP) or platelet‐rich plasma (PRP). PPP and PRP obtained from the same whole blood samples from each of several persons were tested in pairs using the standard Cephotest reagent to observe the consequences of Cephotest being performed on plasma containing platelets. The same procedure was used with a Cephotest reagent with a reduced concentration of phosphatidylserine. In succeeding experiments the PRP was preincubated with SFLLRN or Ca‐ionophore to activate the platelets, a procedure also known to produce platelet‐derived microparticles. PPP and PRP were compared by thrombin time and reptilase time tests to find out at which stage platelets might influence the Cephotest. Results. The results showed that the platelets did influence Cephotest when using both the regular reagent and the phospholipid‐reduced agent. When using the regular reagent, PRP showed a tendency towards a longer APTT than PPP. It is suggested that this was caused by platelets consuming some of the first traces of thrombin generated. Conclusions. When using the phospholipid‐reduced reagent, PRP showed a shorter APTT than PPP, probably because the platelets contributed phosphatidylserine to the system. When the platelets were activated before testing, their effects on the tests were increased. Microparticles that formed during platelet activation may have contributed to these effects.     

Hemostatic effects of fibrinogen gamma-chain dodecapeptide-conjugated polymerized albumin particles in vitro and in vivo

BACKGROUND: Prototypes of platelet (PLT) substitutes have been studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in the fibrinogen domain. STUDY DESIGN AND METHODS: H12 was conjugated to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable particles with a mean diameter of 260 +/- 60 nm, and the hemostatic ability of H12-conjugated polyAlb (H12-polyAlb) under flow conditions and thrombocytopenic rats have been studied. RESULTS: H12-polyAlb enhanced the in vitro thrombus formation of activated PLTs on a collagen-immobilized plate when exposed to the flowing thrombocytopenic imitation blood. Furthermore, the analysis of the tail bleeding time of rats that were made thrombocytopenic by busulfan injection showed that H12-polyAlb had a hemostatic effect. Based on the bleeding time and the amount injected, the hemostatic capacity of 20 H12-polyAlb was estimated to correspond to that of one PLT. CONCLUSION: These results were important first steps toward the development of PLT substitutes and indicated that H12-polyAlb may be a suitable candidate for an alternative to human PLT concentrates transfused into thrombocytopenic patients in the future.     

Differences in platelet function in patients with acute myeloid leukemia and myelodysplasia compared to equally thrombocytopenic patients with immune thrombocytopenia

Summary. Background: Severe thrombocytopenia is a major risk factor for hemorrhage, but platelet function and bleeding risk at low platelet counts are poorly understood, because of the limitations of platelet function testing at very low platelet counts. Objectives: To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukemia (AML) or myelodysplasia (MDS) with that in patients with immune thrombocytopenia (ITP). Methods: Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. Results: Patients with AML/MDS had smaller platelets, lower IPF and substantially lower platelet surface expression of activated glycoprotein (GP)IIb–IIIa and GPIb, both with and without addition of ex vivo ADP or thrombin receptor‐activating peptide, than patients with ITP. In both ITP and AML/MDS patients, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb–IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than for those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than for those with no bleeding. Conclusions: AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP patients. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.     

Reduction of blood loss by infusion of human platelets in a rabbit kidney injury model

BACKGROUND: As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a kidney injury model was developed to assess the hemostatic properties of human platelets in normal and thrombocytopenic rabbits. STUDY DESIGN AND METHODS: New Zealand white rabbits were made thrombocytopenic by two consecutive injections of busulfan. Two weeks later, human platelets were transfused to animals whose reticuloendothelial systems were inhibited by the administration of ethyl palmitate. The left kidney was exposed and a slice excised from the anterior pole. The blood was contained in a parafilm boat and absorbed by preweighed gauze to assess blood loss. The percentage of human platelets transfused to the rabbit was determined by flow cytometry on blood collected from the cut site using anti-CD42a (marker for human platelets). The degree of activation of the human platelets was determined using anti-CD62a (marker specific for human p-selectin). RESULTS: Blood loss was similar in normal animals treated with saline alone (35.4 +/- 5.8 g; n = 4); ethyl palmitate and saline (42.5 +/- 5.7 g; n = 6, p = 0.4); or ethyl palmitate and fresh human platelets (45.7 +/- 7.9 g; n = 6, p = 0.3). Bleeding in thrombocytopenic rabbits infused with saline was increased (75.6 +/- 3.9 g; n = 7) as compared with nonthrombocytopenic animals. A significant reduction in blood loss was noted in thrombocytopenic rabbits given fresh human platelets (51.6 +/- 4.5 g; n = 6, p = 0.0023). Transfusion of human platelets to rabbits did not cause activation of the platelets. Furthermore, transfusion of thrombin-activated platelets (60-98% activated) to thrombocytopenic rabbits reduced blood loss (54 +/- 7.3 g; n = 7) to the same extent as fresh platelets. CONCLUSIONS: This is the first report describing a kidney injury model developed to assess the efficacy of fresh and activated human platelets in reducing blood loss in thrombocytopenic rabbits. This model could monitor the efficacy of human platelets prepared by various preservation protocols in suppressing bleeding in rabbits.     

ADP‐degrading enzymes inhibit platelet activation in bile duct‐ligated rats

Summary. Background: The effect of cholestatic liver disease on primary hemostasis function remains ill‐defined. Objectives: To determine platelet function and identify the mechanisms involved in the observed platelet function in cholestatic rats. Methods: Platelet function was studied in a model of 2‐week bile duct ligation and compared to that in sham‐operated rats with and without a storage pool defect. Results: ADP‐induced and collagen‐induced platelet aggregation were clearly impaired following bile duct ligation (P < 0.01 for areas under the curve). Crossover experiments, with sham platelets in bile duct‐ligated plasma and vice versa, demonstrated that this is due to inhibition by a plasmatic factor, as sham platelets aggregated less in cholestatic plasma (P < 0.03) and to an equal extent as platelets from bile duct‐ligated rats when they were in the same sham or cholestatic plasma. Moreover, in bile duct‐ligated rats, platelet ultrastructure was unaffected and platelet aggregation was similar to that of sham platelets when resuspended in the same plasma (P‐value not significant). Aditionally, studies in storage pool‐deficient rats showed no role of platelet exhaustion. The plasmatic factor causing impaired aggregation was shown to be increased total activity of ADP‐degrading enzymes upon bile duct ligation (P < 0.01), as there was no decreased aggregation with a stable ADP analog in bile duct‐ligated rats (P‐value not significant vs. sham‐operated rats). Furthermore, preincubation of plasma from bile duct‐ligated rats with ADP decreased aggregation more than was seen with sham plasma (P < 0.01). Conclusions: Bile duct ligation does not affect intrinsic platelet function, but impairs platelet activation via release of ADP‐degrading enzymes in the circulation.     

Platelet function normalization after a prasugrel loading‐dose: time‐dependent effect of platelet supplementation

Summary. Background: Hemostatic benefits of platelet transfusions in thienopyridine‐treated acute coronary syndrome (ACS) patients may be compromised by residual metabolite in circulation.Objectives: To estimate the earliest time after a prasugrel loading‐dose when added platelets are no longer inhibited by prasugrel's active metabolite.Methods: Baseline platelet reactivity of healthy subjects (n = 25, 30 ± 5 years, 68% male) on ASA 325 mg was tested using maximum platelet aggregation (MPA, ADP 20 μm ) and VerifyNow^® P2Y12 and was followed by a 60 mg prasugrel loading‐dose. At 2, 6, 12 and 24 h post‐dose, fresh concentrated platelets from untreated donors were added ex‐vivo to subjects’ blood, raising platelet counts by 0% (control), 40%, 60% and 80%. To estimate the earliest time when prasugrel's active metabolite's inhibitory effect on the added platelets ceases, platelet function in supplemented samples was compared across time‐points to identify the time when effect of supplementation on platelet function stabilized (i.e. the increase in platelet reactivity was statistically similar to that at the next time‐point).Results: Supplemented samples showed concentration‐dependent increases in platelet reactivity vs. respective controls by both MPA and VerifyNow^® at all assessment time‐points. For each supplementation level, platelet reactivity showed a sharp increase from 2 to 6 h but was stable (P = NS) between 6 and 12 h.Conclusions: The earliest measured time when supplemented platelets were not inhibited by circulating active metabolite of prasugrel was 6 h after a prasugrel loading‐dose. These findings may have important implications for prasugrel‐treated ACS patients requiring platelet transfusions during surgery.     

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials

• Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP).
• Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF).
• Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model.
• VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Summary

Background

Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS‐13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets.

Objectives

To evaluate the role of obesity and ADAMTS‐13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia.

Methods

Lean or obese ADAMTS‐13‐deficient (Adamts‐13^?/?) and wild‐type (WT) mice were injected with 250 U kg^?1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts‐13^?/? mice by injection of clodronate‐liposomes, 48 h before injection of rVWF.

Results

Obese Adamts‐13^?/? mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts‐13^?/? mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts‐13^?/? mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts‐13^?/? mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose‐dependent increase of platelet phagocytosis.

Conclusions

Obese Adamts‐13^?/? mice are more susceptible to the induction of TTP‐related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

     

Phase I,randomized, double‐blind,placebo‐controlled,single‐dose escalation study of the recombinant factor VIIa variant BAY 86‐6150 in hemophilia

Summary. Background: BAY 86‐6150 is a new human recombinant factor VIIa variant developed for high procoagulant activity and longer action in people with hemophilia with inhibitors. Objectives: To investigate the safety, tolerability, pharmacodynamics, pharmacokinetics and immunogenicity of BAY 86‐6150 in non‐bleeding hemophilia subjects. Methods: The study included non‐bleeding men (18–65 years of age) with moderate or severe hemophilia A or B with or without inhibitors. Sixteen subjects were randomized 3 : 1 to four cohorts of escalating doses of BAY 86‐6150 (6.5, 20, 50 or 90 μg kg^?1 [n = 3 per cohort]) or placebo (n = 1 per cohort); an independent data‐monitoring committee reviewed previous cohort data before the next dose escalation. Blood sampling was performed predose and postdose; subjects were monitored for 50 days postdose. Results: At the tested doses, BAY 86‐6150 was not associated with clinically significant adverse events or dose‐limiting toxicities. BAY 86‐6150 pharmacokinetics exhibited a linear dose response, with a half‐life of 5–7 h. Subjects demonstrated consistent, dose‐dependent thrombin generation ex vivo in platelet‐poor plasma (PPP) (mean peak effect, 26–237 nm thrombin from 6.5 to 90 μg kg^?1). Peak thrombin levels over time paralleled BAY 86‐6150, with thrombin kinetics appearing to be slightly shorter; thus, circulating BAY 86‐6150 retained activity. There were corresponding decreases in activated partial thromboplastin and prothrombin times. No subject developed de novo anti‐BAY 86‐6150 neutralizing antibodies during the 50‐day follow‐up. Conclusions: In this first‐in‐human, multicenter, randomized, double‐blind, placebo‐controlled, single‐dose escalation study, BAY 86‐6150 was tolerated at the highest dose (90 μg kg^?1), with no safety concerns. Safety and efficacy will be further evaluated in phase II/III studies.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Psoralen-mediated photodecontamination of platelet concentrates: inactivation of cell-free and cell-associated forms of human immunodeficiency virus and assessment of platelet function in vivo

BACKGROUND: Treatment of platelet concentrates (PCs) with psoralens and broad-band ultraviolet A (UVA) radiation is being examined for the elimination of pathogens that might be present in donated blood. Previous studies have demonstrated the inactivation of cell-free viruses and the maintenance of platelet integrity with common in vitro assays. STUDY DESIGN AND METHODS: Human immunodeficiency virus (HIV) in three forms-cell-free, activity replicating, and latently infected cell lines-was added to PCs and treated with 50-microgram per mL of 4'- aminomethyl-4,5',8-trimethylpsoralen (AMT), 0.35 mM rutin, and broad- and narrow-band UVA light (320-400 nm and 360–370 nm [UVA1], respectively). The inactivation of added HIV was assessed in tissue culture; platelet hemostatic activity was assessed in thrombocytopenic rabbits. RESULTS: Each form of HIV was inactivated completely (> or = 10(5) infectious units) on treatment with 30 J per cm2 of UVA1 light. Similar results were obtained on treatment of 2.5 mL of PCs in test tubes or intact PC units (50 mL) in blood bags. Latently infected cell lines were substantially more sensitive than cell-free HIV or HIV that was actively replicating. Human platelets treated with 40 J per cm2 of UVA1 light had a fully corrected bleeding time shortly after treatment or after 5 days' storage, as assessed in thrombocytopenic rabbits. Platelet hemostatic function began to decrease with 81 J per cm2 of UVA1 light and was abolished with 113 J per cm2. At similar fluences, broad-band UVA light was more injurious to platelets than was UVA1 light. CONCLUSION: HIV transmission might be eliminated by PCs after treatment with AMT and UVA1 light and without a reduction in platelet hemostatic function.     

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.