von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets

Summary. Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet‐rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin‐free planar fibrin layers and washed platelets to examine the relationship between platelet–fibrin interaction and exposure of coagulation‐stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s^?1 requires fibrin‐bound VWF. The cytosolic calcium concentration ([Ca²⁺]_i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 ± 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 ± 14%) along with a transient elevation in [Ca²⁺]_i from 0.05 µmol L^?1 up to 1.1 ± 0.2 µmol L^?1. Platelet adhesion to fibrin at a shear rate of 50 s^?1 is mediated by thrombin but not by fibrin‐bound VWF. The [Ca²⁺]_i of these thrombin‐activated platelets was elevated (0.2 ± 0.1 µmol L^?1), but only a minority of the platelets (11 ± 8%) exposed PS. The essential role of VWF in this thrombin‐induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 ± 23%) exposed PS and had peak values of [Ca²⁺]_i of about 0.6 µmol L^?1. Taken together, these results demonstrate that thrombin‐induced exposure of PS and high calcium response on fibrin‐adherent platelets depends on shear‐ or botrocetin‐induced VWF–platelet interaction.     

Differential contributions of monocyte‐ and platelet‐derived microparticles towards thrombin generation and fibrin formation and stability

Summary. Background: Microparticles (MPs) are sub‐micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood. Objective: To determine how MPs promote thrombin generation and modulate fibrin density and stability. Methods: Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP‐1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high‐speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability. Results: MPs from monocytes (M‐MPs) and platelets (PMPs) had similar shapes and diameters (100–300 nm). M‐MPs had TF activity (16.7 ± 2.4 pm TF per 10⁶ MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M‐MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min^?1, respectively), 38% higher fibrin network density and higher clot stability (3.8‐fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8‐fold lower prothrombinase activity than M‐MPs. PMPs supported contact‐dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7‐ and 1.3‐fold, respectively) when mixed with THP‐1‐derived MPs. Conclusion: MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.     

Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding

Summary. Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue‐factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin‐mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin‐dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti‐TF antibodies inhibiting TF‐induced thrombin formation in platelet‐depleted plasma had no effect compared with vehicle‐treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase‐inhibitors bacitracin and anti‐PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin‐stimulated platelets exhibited increased surface‐associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin‐stimulated platelets. Conclusions: Thus, surface‐associated PDI is an important regulator of coagulation factor ligation to thrombin‐stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.     

Diannexin,an annexin A5 homodimer,binds phosphatidylserine with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet‐mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS‐containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet‐fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser‐induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with K_D values of 0.6 and 5 nm , respectively, and both bind to the same subpopulation of PS‐exposing platelets. Diannexin inhibited thrombin generation and platelet‐fibrin clot formation in vitro at 10 nm (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 μg g^?1 (P < 0.05) and activation at 0.25 μg g^?1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g^?1 (P < 0.01). Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation.     

Platelet‐targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse

Summary. Background: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. Methods and results: An N‐terminal‐azido thrombin‐sensitive fluorescent peptide (ThS‐P) with a thrombin‐releasable quencher was linked to anti‐CD41 using click chemistry to generate a thrombin‐sensitive platelet binding sensor (ThS‐Ab). Rapid thrombin cleavage of ThS‐P (K_m = 40.3 μm , k_cat = 1.5 s^?1) allowed thrombin monitoring by ThS‐P or ThS‐Ab in blood treated with 2–25 pm tissue factor (TF). Individual platelets had > 20‐fold more ThS‐Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s^?1), ThS‐Ab fluorescence increased between 90 and 450 s for 0.1–1 molecule‐TF μm^?2 and co‐localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s^?1), thrombin and fibrin were detected at the thrombus–collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti‐mouse CD41 ThS‐Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co‐localized and where the thrombus was most mechanically stable. Conclusion: ThS‐Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.     

Platelet membrane glycoproteins in haemostasis

Platelets play a critical role in both primary and secondary haemostasis. In primary haemostasis, specialised glycoprotein receptors enable platelets to adhere to proteins that are exposed in areas of vascular damage. The process of adhesion, and/or the interaction of soluble agonists with receptors on the platelet, activates the platelets, which are then able to aggregate together. This aggregation creates a platelet plug that seals the breach in the vessel wall and prevents excess blood loss. Activated platelets then facilitate secondary haemostasis, the formation of a fibrin clot, by carrying coagulation factors and providing a catalytic surface for the major interactions of the coagulation cascade. This review briefly describes the adhesion, activation and aggregation of platelets; their role in blood coagulation and clot retraction; and how they may be inhibited in order to prevent thrombosis in at-risk patients.     

A high affinity,antidote‐controllable prothrombin and thrombin‐binding RNA aptamer inhibits thrombin generation and thrombin activity

Background:  The conversion of prothrombin to thrombin is one of two non‐duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti‐thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D‐14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. Objectives: We sought to explore the structure of R9D‐14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA_R9D‐14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results:  RNA_R9D‐14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I‐mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA_R9D‐14T significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC‐183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA_R9D‐14T anticoagulation in vitro. Conclusions:  Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA_R9D‐14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.     

Factor XIII cotreatment with hemostatic agents in hemophilia A increases fibrin α‐chain crosslinking

Essentials

• Factor XIII (FXIII)‐mediated fibrin crosslinking is delayed in hemophilia.
• We determined effects of FXIII cotreatment with hemostatic agents on clot parameters.
• FXIII cotreatment accelerated FXIII activation and crosslinking of fibrin and α₂‐antiplasmin.
• These data provide biochemical rationale for FXIII cotreatment in hemophilia.

Summary

Background

Hemophilia A results from the absence, deficiency or inhibition of factor VIII. Bleeding is treated with hemostatic agents (FVIII, recombinant activated FVII [rFVIIa], anti‐inhibitor coagulation complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment, some patients have prolonged bleeding. FXIII‐A₂B₂ (FXIII) is a protransglutaminase. During clot contraction, thrombin‐activated FXIII (FXIIIa) crosslinks fibrin and α₂‐antiplasmin, which promotes red blood cell retention and increases clot stability and weight. We hypothesized that FXIII cotreatment in hemophilia would accelerate FXIII activation, leading to increased fibrin crosslinking.

Methods

FVIII‐deficient plasma and whole blood were clotted with or without hemostatic agents (FVIII, rFVIIa, FEIBA, or recombinant B‐domain‐deleted porcine FVIII [rpFVIII]) and/or FXIII. The effects on FXIII activation, thrombin generation, fibrin and α₂‐antiplasmin crosslinking, clot formation and clot weight were measured by western blotting, calibrated automated thrombography, thromboelastography, and clot contraction assays.

Results

As compared with FVIII‐treated hemophilic plasma, FVIII + FXIII cotreatment accelerated FXIIIa formation without increasing thrombin generation. As compared with buffer‐treated or FXIII‐treated hemophilic plasma, FVIII treatment and FVIII + FXIII cotreatment increased the generation and amount of crosslinked fibrin, including α‐chain‐rich high molecular weight species and crosslinked α₂‐antiplasmin. In the presence of FVIII inhibitors, as compared with hemostatic treatments (rFVIIa, FEIBA, or rpFVIII) alone, FXIII cotreatment increased whole blood clot weight.

Conclusion

In hemophilia A plasma and whole blood, FXIII cotreatment with hemostatic agents accelerated FXIIIa formation, increased the generation and amount of fibrin α‐chain crosslinked species, accelerated α₂‐antiplasmin crosslinking, and increased clot weight. FXIII cotreatment with hemostatic therapy may augment hemostasis through increased crosslinking of fibrin and α₂‐antiplasmin.     

Platelets loaded with liposome‐encapsulated thrombin have increased coagulability

Essentials

• Platelet transfusions can have limited efficacy during hemorrhage associated with coagulopathy.
• Thrombin can be shielded by encapsulation into nanoliposomes and delivered to platelets ex vivo.
• Loading platelets with liposomal thrombin improved several aspects of platelet coagulability.
• Platelets loaded with liposomal thrombin can overcome some coagulopathic deficiencies in vitro.

Summary

Background

Platelets are integral to clot formation and are often transfused to stop or prevent bleeding. However, transfusions of platelets are not always effective, particularly in the most severe cases of hemorrhage. Nanoparticle systems have been developed to mimic platelets but inherently lack important aspects of platelet function, which limits their potential effectiveness.

Objectives

Increasing the natural coagulability of transfusable platelets could increase their efficacy during treatment of severe hemorrhage. Thrombin is a potent platelet agonist that currently cannot be used intravenously because of the risk of thrombosis. We hypothesized that delivery of thrombin to ex vivo platelets via liposomal encapsulation would enable transfusable platelets to become more coagulable in response to platelet agonists.

Methods

Thrombin was encapsulated into nanoliposomes and delivered to platelets ex vivo. Platelet coagulability was measured by monitoring platelet activation, clot contraction, clot time and clot stability in several in vitro assays. These parameters were also measured under conditions where coagulation is compromised, including during acidosis, antiplatelet drugs, hemophilia A and trauma‐induced coagulopathy.

Results

Liposomal thrombin was endocytosed and used by platelets ex vivo but was not secreted upon activation. These modified platelets became more sensitive and responsive to agonists and improved clotting time even under conditions that normally cause platelet dysfunction or have impaired coagulation.

Conclusions

Several aspects of platelet function were enhanced by ex vivo delivery of liposomal thrombin.

     

Procoagulant platelets: Generation,characteristics, and therapeutic target

Platelets play a pivotal role in hemostasis. Activated platelets are classified into two groups, according to their agonist response: aggregating and procoagulant platelets. Aggregating platelets consist of activated integrin αIIbβ3 and stretch out pseudopods to further attract platelets to the site of injury by connecting with fibrinogen. They mainly gather in the core of the thrombus and perform a secretory function, such as releasing adenosine diphosphate (ADP). Procoagulant platelets promote the formation of thrombin and fibrin by interacting with coagulation factors and can thus be considered as the connector between primary and secondary hemostasis. In addition to their functions in blood coagulation, procoagulant platelets play a proinflammatory role by releasing platelet microparticles and inorganic polyphosphate. Considering these important functions of procoagulant platelets, this subpopulation warrants detailed study to analyze their potential in preventing human diseases. This review summarizes the generation and important characteristics of procoagulant platelets, as well as their potential for preventing the adverse effects associated with current antiplatelet therapies.     

Transport physics and biorheology in the setting of hemostasis and thrombosis

The biophysics of blood flow can dictate the function of molecules and cells in the vasculature with consequent effects on hemostasis, thrombosis, embolism, and fibrinolysis. Flow and transport dynamics are distinct for (i) hemostasis vs. thrombosis and (ii) venous vs. arterial episodes. Intraclot transport changes dramatically the moment hemostasis is achieved or the moment a thrombus becomes fully occlusive. With platelet concentrations that are 50‐ to 200‐fold greater than platelet‐rich plasma, clots formed under flow have a different composition and structure compared with blood clotted statically in a tube. The platelet‐rich, core/shell architecture is a prominent feature of self‐limiting hemostatic clots formed under flow. Importantly, a critical threshold concentration of surface tissue factor is required for fibrin generation under flow. Once initiated by wall‐derived tissue factor, thrombin generation and its spatial propagation within a clot can be modulated by γ′‐fibrinogen incorporated into fibrin, engageability of activated factor (FIXa)/activated FVIIIa tenase within the clot, platelet‐derived polyphosphate, transclot permeation, and reduction of porosity via platelet retraction. Fibrin imparts tremendous strength to a thrombus to resist embolism up to wall shear stresses of 2400 dyne cm^?2. Extreme flows, as found in severe vessel stenosis or in mechanical assist devices, can cause von Willebrand factor self‐association into massive fibers along with shear‐induced platelet activation. Pathological von Willebrand factor fibers are A Disintegrin And Metalloprotease with ThromboSpondin‐1 domain 13 resistant but are a substrate for fibrin generation due to FXIIa capture. Recently, microfluidic technologies have enhanced the ability to interrogate blood in the context of stenotic flows, acquired von Willebrand disease, hemophilia, traumatic bleeding, and drug action.     

Patients with Congenital Factor V Deficiency have Decreased Factor Xa Binding Sites on their Platelets

Human platelets have binding sites for plasma coagulation Factor X_a that are available only after the platelet release reaction. Platelets from 15 normal donors bound 216±52 (SD) molecules of Factor X_a per platelet. The association of Factor X_a with its platelet surface receptor results in a 300,000-fold increase in the catalytic activity of Factor X_a in forming thrombin from prothrombin. The turnover number for platelet-bound Factor X_a was 1,850±460 mol thrombin/ml per min per mol Factor X_a in experiments with platelets from 15 normal donors. Platelets from five patients with varying degrees of Factor V deficiency were investigated to determine whether or not coagulation Factor V participates in either aspect of the Factor X_a-platelet interaction. The binding of Factor X_a to platelets and the accompanying increase in rate of thrombin formation were either reduced in parallel or absent in each case with values ranging from 0 to 45% of control values. The apparent affinity of Factor X_a from Factor V-deficient patients was normal when platelet binding was detected. The supernate from thrombin-treated control platelets, which contains Factor V activity, corrected the Factor X_a binding deficiency of the platelets from three patients tested. Immunoreactive Factor V determined with an homologous antibody corresponded to the functional Factor V activity of platelets from one patient with Factor V deficiency, suggesting that the patient's platelets have a decreased amount of normal Factor V.     

Platelet‐ and erythrocyte‐derived microparticles trigger thrombin generation via factor XIIa

See also Shapiro S, Laffan M. Making contact with microparticles. This issue, pp 1352–4. Summary. Background: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. Objectives: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. Methods: Microparticles were isolated from calcium ionophore‐stimulated platelets, erythrocytes and monocytic THP‐1 cells. MPs were quantified, characterized for cell‐specific antigens and analyzed for TF, PS exposure and their thrombin‐generating potential. Results: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet‐ and erythrocyte‐derived MPs (< 1 fm nm ^?1 PS), whereas monocyte‐derived MPs exposed TF (32 fm nm ^?1 PS). Platelet‐, erythrocyte‐ and monocyte‐derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet‐ and erythrocyte‐derived MPs, but interfered with monocyte MP‐triggered coagulation. Platelet‐ and erythrocyte‐derived MPs completely failed to induce thrombin generation in FXII‐deficient plasma. In contrast, monocyte‐derived MPs induced similar thrombin generation in normal vs. FXII‐deficient plasma. Conclusion: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII‐dependent manner. In contrast, monocyte‐derived MPs trigger coagulation predominantly via TF.     

Clot retraction: evaluation in dilute suspensions of platelet-rich plasma and gel-separated platelets.

Suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP) can be used to evaluate clot retraction subsequent to platelet aggregation and fibrin formation. PRP (200,000 per cubic millimeter) or GSP (200,000 or 100,000 per cubic millimeter) are diluted 1:10 (PRP) or 1:8 (GSP) in phosphate buffer, pH 7.4, and clotted with a high concentration (2.5 U. per milliliter) of thrombin. Human fibrinogen (25 mg. per cent) is added to GSP prior to dilution. Clot retraction is 91 to 100 per cent completed in 1 hour and is quantified by measurement of residual fluid volume. Test conditions are unfavorable for fibrinolysis. Very low concentrations of fibrin/fibrinogen degradation products D and E are detected in residual fluid, and no erythrocyte fall-out occurs. Furthermore, the extent of retraction in the dilute systems is related only to platelet numbers and platelet function. The dilute PRP and GSP methods allow evaluation of clot retraction in the presence of PGE1, the most potent inhibitor of platelet aggregation induced by conventional concentrations of collagen, ADP, epinephrine, and thrombin (0.1 to 0.5 U. per milliliter). High concentrations of PGE1 (to 6 x 10(-6) M) do not inhibit aggregation of GSP, fibrin formation, or platelet-fibrin interaction induced by 2.5 U. per milliliter of thrombin. In contrast, PGE1 concentrations as low as 1.5 to 3.0 x 10(-8) M inhibit clot retraction in both the dilute PRP and GSP systems. Thus, using dilute PRP or GSP the effects of platelet aggregation inhibitors on clot retraction can be determined independently of effects on platelet aggregation.     

Relative contributions of stromal interaction molecule 1 and CalDAG‐GEFI to calcium‐dependent platelet activation and thrombosis

Summary. Background: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store‐operated calcium entry (SOCE) in platelets. We previously reported the Ca²⁺‐sensing guanine nucleotide exchange factor CalDAG‐GEFI as a critical molecule in Ca²⁺ signaling in platelets. Objective: To evaluate the contribution of STIM1/SOCE to Ca²⁺‐dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG‐GEFI. Methods: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG‐GEFI^–/– and Stim1^fl/flPF4‐Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. Results: Integrin α_IIbβ₃‐mediated aggregation was markedly impaired in CalDAG‐GEFI‐deficient but not STIM1‐deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG‐GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet‐rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG‐GEFI^–/– mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. Conclusions: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG‐GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets.     

Platelets and the new comprehension of haemostasis

Platelets are cells with key function in primary haemostasis. They localise coagulation to the haemostatic thrombus. After injury of the vessel wall blood contacts subendothelial matrix proteins as well as cells constitutively exposing tissue factor (TF). Platelets adhere to the subendothelial matrix, become activated, spread and secrete the contents of their granules. On the surface of the TF exposing cells minute amounts of thrombin are formed. These amounts of thrombin are inadequate to yield in a stable fibrin clot, but activate platelets and factors XI, VIII, V. In that way the consolidation pathway is triggered. Activated platelets aggregate and bind leukocytes. On the surface of the activated platelets coagulation (co)factor complexes are formed and protected in an optimal way. Thus large amounts of prothrombin are converted to thrombin, creating a so-called thrombin burst. This leads to the formation of a stable platelet-fibrin-clot. Platelets are not always prothrombotic. They have their own mechanisms to stop activation processes and thrombus growth. Besides, its key role in haemostasis platelets are involved in inflammation and innative immune defence.     

Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT)

Summary.  Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin–antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.     

Qualitative description of factors involved in the retraction and lysis of dilute whole blood clots and in the aggregation and retraction of platelets

Dilute whole blood clots were prepared by addition of thrombin to blood diluted 1:10 in phosphate buffer. The pH of this buffer was 7.4 and the ionic strength was 0.084. Though the ionic strength was low, there was no hemolysis of red corpuscles due to the contribution to the osmotic gradient by plasma salts and proteins. In the standard assay the clot was formed by addition of thrombin at 4 degrees C then incubated at 37 degrees C. Retraction and lysis of these clots were inhibited by removal of platelets and by increasing concentrations of purified thrombin. Retraction and lysis were also inhibited by inactivation of any one of the following factors: gammaM globulin, complement components C4 and 3, and (in the case of lysis) plasminogen.Evidence that some of the above serum factors were adsorbed to the platelet membrane was obtained by aggregation of washed platelets by antisera to these factors (i.e. fibrinogen, gammaM, and C4 or C3). These platelets were not aggregated by antisera to other serum proteins (by albumin, transferrin, gammaG globulin).These and other studies suggested that platelets, thrombin, fibrinogen, gammaM globulin (cold agglutinin), complement components, and plasminogen influenced and facilitated retraction and lysis of clots. These studies also suggested that platelets and some of these factors were physically associated.Because of this physical association, and because of the fact that clot retraction is associated with aggregation and retraction of platelets, we extended the above observations to include a study of the effect of these same serum factors on serum-induced aggregation and retraction of washed platelets. (Other terms which have been in use in the past to describe serum-induced platelet aggregation and retraction have included those such as platelet "fusion" and "viscous metamorphosis," neither of which fully described the phenomena.)Platelet aggregation and retraction induced by serum was markedly accelerated by addition of increasing concentrations of thrombin and (or) cold agglutinin. Hirudin and antisera to gammaM globulin inhibited seruminduced aggregation and retraction of platelets. Reconstitution of inactivated serum with purified C4, 3, and 5 and thrombin restored its capacity to induce aggregation and retraction of platelets.Therefore, we postulated that platelet aggregation and retraction were necessary for clot retraction and that platelet aggregation and clot retraction facilitated clot lysis. More specifically we postulated that thrombin, in addition to catalyzing clot formation, also modified the platelet membrane such that gammaM globulin (cold agglutinin) and complement components can act on the platelet membrane leading to (a) aggregation and retraction of the platelets, (b) retraction of the clot, and (c) to the activation of plasminogen either on the surface of the platelet by C8i and (or) by release of platelet activators of plasminogen.     

Thrombin activatable fibrinolysis inhibitor (TAFI)—How does thrombin regulate fibrinolysis?

The thrombin‐catalysed conversion of plasma fibrinogen into fibrin and the development of an insoluble fibrin clot are the final steps of the coagulation cascade during haemostasis. A delicate balance between coagulation and fibrinolysis determines the stability of the fibrin clot. Thrombin plays a central role in this process, it not only forms the clot but it is also involved in stabilizing the clot by activating thrombin activatable fibrinolysis inhibitor (TAFI). Activated TAFI protects the fibrin clot against lysis. Here we will discuss the mechanisms for regulation of fibrinolysis by thrombin. The role of the coagulation system for the generation of thrombin and for the activation of TAFI implies that defects in thrombin generation will directly affect the protection of clots against lysis. Thus, defects in activation of TAFI might contribute to the severity of bleeding disorders. Vice versa an increased activation of TAFI due to an increased rate of thrombin generation might lead to thrombotic disorders. Specific inhibitors of activated TAFI or inhibitors that interfere with the generation of thrombin might provide novel therapeutic strategies for thrombolytic therapy. Besides having a role in the regulation of fibrinolysis, TAFI may also have an important function in the regulation of inflammation, wound healing and blood pressure.     

Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibition

Summary. Background: Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non‐diabetic subjects. Objective: We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Methods: Patients with T2DM and gender/age/body mass index‐matched non‐diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet‐derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet‐dependent thrombin generation and plasma clotting time were monitored in recalcified platelet‐rich plasma. Results: Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Conclusions: Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade.