Phosphatidylserine-exposing Platelets Research Articles

Quantitation of phosphatidylserine-exposing platelets and platelet-derived microparticles in platelet products: A new strategy to improve efficacy of platelet transfusion

Platelet transfusion is an effective therapy to prevent or treat bleeding. Considering the different clinical purposes of transfusion, it is necessary to assess the quality of platelet products prepared in transfusion laboratories. So far, there is no solution to the problem of how best to do this. Here, we summarize the quantitation of phosphatidylserine (PS)-exposing platelets and platelet-derived microparticles (PMPs) in platelet products using previously reported data. Because PS promotes the assembly and enhances the activity of coagulation factors, classifying platelet products according to their concentrations of PS-exposing platelets and PMPs will improve the therapeutic treatment of transfusion recipients.

Functional plasminogen activator inhibitor 1 is retained on the activated platelet membrane following platelet activation.

Platelets harbor the primary reservoir of circulating plasminogen activator inhibitor 1 (PAI-1), but the reportedly low functional activity of this pool of inhibitor has led to debate over its contribution to thrombus stability. Here we analyze the fate of PAI-1 secreted from activated platelets and examine its role in maintaining thrombus integrity. Activation of platelets results in translocation of PAI-1 to the outer leaflet of the membrane, with maximal exposure in response to strong dual agonist stimulation. PAI-1 is found to co-localize in the 'cap' of phosphatidylserine-exposing platelets with its co-factor, vitronectin, and fibrinogen. Inclusion of tirofiban or Gly-Pro-Arg-Pro significantly attenuated exposure of PAI-1, indicating a crucial role for integrin αIIbβ3 and fibrin in delivery of PAI-1 to the activated membrane. Separation of platelets post stimulation into soluble and cellular components revealed the presence of PAI-1 antigen and activity in both fractions, with approximately 40% of total platelet-derived PAI-1 remaining associated with the cellular fraction. Using a variety of fibrinolytic models, we found that platelets produce a strong stabilizing effect against tissue plasminogen activator (tPA)-mediated clot lysis. Platelet lysate, as well as soluble and cellular fractions, stabilize thrombi against premature degradation in a PAI-1-dependent manner. Our data show for the first time that a functional pool of PAI-1 is anchored to the membrane of stimulated platelets and regulates local fibrinolysis. We reveal a key role for integrin αIIbβ3 and fibrin in delivery of PAI-1 from platelet α-granules to the activated membrane. These data suggest that targeting platelet-associated PAI-1 may represent a viable target for novel profibrinolytic agents.

Quantitation of phosphatidylserine-exposing platelets in platelet concentrate prepared in routine blood transfusion laboratory

BackgroundPhosphatidylserine (PS) plays important roles in platelets’ pro-coagulant function. However, little is known about assessing this molecule in platelet concentrates (PCs) prepared for routine blood transfusion service. AimTo quantitate the number of PS-exposing platelets in PCs prepared in a routine transfusion laboratory. MethodsPC products were prepared according to routine laboratory procedure. The numbers of PS-exposing platelets in the PCs and in unprocessed whole blood were determined using flow cytometry. ResultsA cross-sectional study of 253 PCs found that they had significantly increased numbers of PS-exposing platelets compared to unprocessed whole blood (47,439 ± 26,500 cells/μL; 5903‒166,156 cells/μL) vs. 30,058 ± 12,958 cells/μL; 8,154-86,606 cells/μL). A heterogeneity study demonstrated that 6% and 2% of the measured PCs and of unprocessed donor whole blood, respectively, showed an increase in the number of PS-exposing platelets that was greater than 2 fold. ConclusionsThe study suggested that the number PS-exposing platelets in PC prepared in a routine transfusion laboratory differs. However, assessment of the number of PS-exposing platelets in platelet products could be a valid measure to use in managing the quality of platelet processing in routine laboratories.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII.

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)β3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)β3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)β3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)β3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)β3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)β3.

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

AbstractThe interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow.

The importance of factor Xa generation in thrombus formation has not been studied extensively so far. Here, we used mice deficient in either factor VIII or factor IX to determine the role of platelet-stimulated tenase activity in the formation of platelet-fibrin thrombi on collagen. With tissue factor present, deficiency in factor VIII or IX markedly suppressed thrombus growth, fibrin formation and platelet procoagulant activity (phosphatidylserine exposure). In either case, residual fibrin formation was eliminated in the absence of tissue factor. Effects of factor deficiencies were antagonized by supplementation of the missing coagulation factor. In wild-type thrombi generated under flow, phosphatidylserine-exposing platelets bound (activated) factor IX and factor X, whereas factor VIII preferentially co-localized at sites of von Willebrand factor binding. Furthermore, proteolytic activity of the generated activated factor X and thrombin was confined to the sites of phosphatidylserine exposure. With blood from a hemophilia A or B patient, the formation of platelet-fibrin thrombi was greatly delayed and reduced, even in the presence of high concentrations of tissue factor. A direct activated factor X inhibitor, rivaroxaban, added to human blood, suppressed both thrombin and fibrin formation. Together, these data point to a potent enforcement loop in thrombus formation due to factor X activation, subsequent thrombin and fibrin generation, causing activated factor X-mediated stimulation of platelet phosphatidylserine exposure. This implies that the factor VIII/factor IX-dependent stimulation of platelet procoagulant activity is a limiting factor for fibrin formation under flow conditions, even at high tissue factor concentrations.

Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Dual role of collagen in factor XII–dependent thrombus formation

AbstractIn vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

Key Role of Platelet Procoagulant Activity in Tissue Factor‐and Collagen‐Dependent Thrombus Formation in Arterioles and Venules In Vivo Differential Sensitivity to Thrombin Inhibition

Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation. Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels. Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation. In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.

Segregation of Platelet Aggregatory and Procoagulant Microdomains in Thrombus Formation

Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated alphaIIb beta3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active alphaIIb beta3. These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication.