Identification of platelet function defects by multi-parameter assessment of thrombus formation

B, respectively, causes a mild to severe bleeding disorder. Early work, particularly using animal models, indicated that in these forms of hemophilia initial platelet plug formation (primary hemostasis) is unaffected, whereas ensuing fibrin formation (secondary hemostasis) is markedly diminished. [22][23][24] This can be explored in test-tube experiments, suggesting that certain rates of FXa as well as thrombin generation are needed to produce a stable hemostatic platelet-fibrin clot, and that both FVIII and FIX are required for sufficient levels of FXa generation.

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“…37 Samples of citrate-anticoagulated blood (400-1500 μL) were made to flow at defined wall-shear rate, as described for murine 38 …”

Section: Whole Blood Flow Chamber Experimentsmentioning

confidence: 99%

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

et al. 2015

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“…Furthermore, DiOC 6 -labeled platelet volume was investigated using confocal z-stacks (16-bit images of 1024 3 1024 pixels; 336 3 336 mm; stack distance 0.5 mm; 100 slices). 42 No difference was noted in total platelet volume 6 75 nM tPA (311 mm 3 6 145 mm 3 vs 267 mm 3 6 115 mm 3 per field, P 5 .43, n 5 4). Differences in fibrinolysis were noted in cardiothoracic surgery patients treated with 2 mg of tranexamic acid, a lysine analog that inhibits plasminogen activation.…”

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confidence: 99%

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

Whyte

Swieringa

Mastenbroek

et al. 2015

Blood

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Key Points• Under physiological flow rates, plasminogen primarily accumulates on fibrin(ogen), emanating from platelets and initiates fibrinolysis.• Plasminogen is localized to defined "caps" on the surface of PS-exposing platelets in a fibrin(ogen)-dependent manner.The 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 GlyPro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PSexposing 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. (Blood. 2015;125(16):2568-2578 IntroductionPlatelet accumulation is central to the hemostatic response. Platelets are activated in vivo by numerous agonists of varying potency, including thrombin, collagen, adenosine 59diphosphate, and thromboxane A2. Platelets exhibit a nonuniform response to activation, with distinct populations forming with different surface characteristics.1 Aggregating platelets are characterized by a spherical shape, binding of fibrinogen, and expression of the active integrin a IIb b 3, and predominantly function in clot retraction. Highly activated platelets are observed on collagen fibers 1 and in the core region of a thrombus nearest the vascular injury.2 These platelets are characterized by membrane exposure of phosphatidylserine (PS), a rounded balloon-like structure, sustained increase in cytosolic Ca 21, and binding of coagulation factors. 3,4 PS-exposing platelets, also termed procoagulant platelets, substantially enhance the activity of the prothrombinase complex, 5,6 and subsequent thrombin and fibrin formation. 7 An additional subpopulation of platelets, termed "coated" platelets, are generated in response to strong dual agonist stimulatio...

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“…1,2 In a growing thrombus, aggregated and procoagulant platelets form two distinct populations, 3,4 which is at least partly explained by the high Ca 2+ response required for PS exposure and coagulation factor binding, and by the calpain-dependent closure of active α IIb b 3 integrins after PS exposure, thus antagonizing inclusion of procoagulant platelets into a platelet aggregate. 5,6 However, another platelet population has also been identified, usually referred to as coated platelets, 7 which may partly overlap with the two other platelet populations described above.…”

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Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

et al. 2015

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C oated 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 b 3 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 b 3 . 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 b 3 . Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α IIb b 3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α IIb b 3 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 b 3 .

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Identification of platelet function defects by multi-parameter assessment of thrombus formation

Cited by 197 publications

References 53 publications

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

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

Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII

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Identification of platelet function defects by multi-parameter assessment of thrombus formation

Cited by 197 publications

References 53 publications

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

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

Coated platelets function in platelet-dependent fibrin formation via integrin α IIb β 3 and transglutaminase factor XIII

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Coated platelets function in platelet-dependent fibrin formation via integrin α _IIb β ₃ and transglutaminase factor XIII