S100A8/A9 drives the formation of procoagulant platelets through GPIbα.

S100A8/A9, also known as "calprotectin" or "MRP8/14," is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo.

Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

Past, Present, and Future Perspectives of Plasminogen Activator Inhibitor 1 (PAI-1).

Plasminogen activator inhibitor 1 (PAI-1), a SERPIN inhibitor, is primarily known for its regulation of fibrinolysis. However, it is now known that this inhibitor functions and contributes to many (patho)physiological processes including inflammation, wound healing, cell adhesion, and tumor progression.This review discusses the past, present, and future roles of PAI-1, with a particular focus on the discovery of this inhibitor in the 1970s and subsequent characterization in health and disease. Throughout the past few decades diverse functions of this serpin have unraveled and it is now considered an important player in many disease processes. PAI-1 is expressed by numerous cell types, including megakaryocytes and platelets, adipocytes, endothelial cells, hepatocytes, and smooth muscle cells. In the circulation PAI-1 exists in two pools, within plasma itself and in platelet α-granules. Platelet PAI-1 is secreted following activation with retention of the inhibitor on the activated platelet membrane. Furthermore, these anucleate cells contain PAI-1 messenger ribonucleic acid to allow de novo synthesis.Outside of the traditional role of PAI-1 in fibrinolysis, this serpin has also been identified to play important roles in metabolic syndrome, obesity, diabetes, and most recently, acute respiratory distress syndrome, including coronavirus disease 2019 disease. This review highlights the complexity of PAI-1 and the requirement to ascertain a better understanding on how this complex serpin functions in (patho)physiological processes.

Exposure of plasminogen and a novel plasminogen receptor, Plg-RKT, on activated human and murine platelets.

Plasminogen activation rates are enhanced by cell surface binding. We previously demonstrated that exogenous plasminogen binds to phosphatidylserine-exposing and spread platelets. Platelets contain plasminogen in their α-granules, but secretion of plasminogen from platelets has not been studied. Recently, a novel transmembrane lysine-dependent plasminogen receptor, Plg-RKT, has been described on macrophages. Here, we analyzed the pool of plasminogen in platelets and examined whether platelets express Plg-RKT. Plasminogen content of the supernatant of resting and collagen/thrombin-stimulated platelets was similar. Pretreatment with the lysine analog, ε-aminocaproic acid, significantly increased platelet-derived plasminogen (0.33 vs 0.08 nmol/108 platelets) in the stimulated supernatant, indicating a lysine-dependent mechanism of membrane retention. Lysine-dependent, platelet-derived plasminogen retention on thrombin and convulxin activated human platelets was confirmed by flow cytometry. Platelets initiated fibrinolytic activity in fluorescently labeled plasminogen-deficient clots and in turbidimetric clot lysis assays. A 17-kDa band, consistent with Plg-RKT, was detected in the platelet membrane fraction by western blotting. Confocal microscopy of stimulated platelets revealed Plg-RKT colocalized with platelet-derived plasminogen on the activated platelet membrane. Plasminogen exposure was significantly attenuated in thrombin- and convulxin-stimulated platelets from Plg-RKT-/- mice compared with Plg-RKT+/+ littermates. Membrane exposure of Plg-RKT was not dependent on plasminogen, as similar levels of the receptor were detected in plasminogen-/- platelets. These data highlight Plg-RKT as a novel plasminogen receptor in human and murine platelets. We show for the first time that platelet-derived plasminogen is retained on the activated platelet membrane and drives local fibrinolysis by enhancing cell surface-mediated plasminogen activation.

Atorvastatin-mediated inhibition of prenylation of Rab27b and Rap1a in platelets attenuates their prothrombotic capacity and modulates clot structure.

Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.

What is the context? Statins such as Atorvastatin (ATV) are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol.The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases.Prenyl groups provide a membrane anchor that is essential for the correct membrane localization and function of these proteins.Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins.Rab27b and Rap1 are small GTPase proteins in platelets that are involved in the secretion of platelet granules and integrin activation.What is new?In this study, we found that ATV impairs prenylation of Rab27b and Rap1a and attenuates platelet function.These effects were partially rescued by GGPP, indicating the involvement of the mevalonate pathway.Platelet aggregation and degranulation was significantly attenuated by ATV.The impact of statins on platelet function altered clot formation, structure and contraction generating a clot that was more susceptible to degradation.What is the impact?This study demonstrates a novel mechanism whereby statins alter platelet responses and ultimately clot structure and stability.

Cellular FXIII in Human Macrophage-Derived Foam Cells.

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery.

The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.

Cryoprecipitate transfusion in trauma patients attenuates hyperfibrinolysis and restores normal clot structure and stability: Results from a laboratory sub-study of the FEISTY trial.

BACKGROUND: Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. METHODS: Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. RESULTS: Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. CONCLUSIONS: In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

The Efficacy of Fibrinogen Concentrates in Relation to Cryoprecipitate in Restoring Clot Integrity and Stability against Lysis.

Loss of fibrinogen is a feature of trauma-induced coagulopathy (TIC), and restoring this clotting factor is protective against hemorrhages. We compared the efficacy of cryoprecipitate, and of the fibrinogen concentrates RiaSTAP® and FibCLOT® in restoring the clot integrity in models of TIC. Cryoprecipitate and FibCLOT® produced clots with higher maximal absorbance and enhanced resistance to lysis relative to RiaSTAP®. The fibrin structure of clots, comprising cryoprecipitate and FibCLOT®, mirrored those of normal plasma, whereas those with RiaSTAP® showed stunted fibers and reduced porosity. The hemodilution of whole blood reduced the maximum clot firmness (MCF) as assessed by thromboelastography. MCF could be restored with the inclusion of 1 mg/mL of fibrinogen, but only FibCLOT® was effective at stabilizing against lysis. The overall clot strength, measured using the Quantra® hemostasis analyzer, was restored with both fibrinogen concentrates but not cryoprecipitate. α2antiplasmin and plasminogen activator inhibitor-1 (PAI-1) were constituents of cryoprecipitate but were negligible in RiaSTAP® and FibCLOT®. Interestingly, cryoprecipitate and FibCLOT® contained significantly higher factor XIII (FXIII) levels, approximately three-fold higher than RiaSTAP®. Our data show that 1 mg/mL fibrinogen, a clinically achievable concentration, can restore adequate clot integrity. However, FibCLOT®, which contained more FXIII, was superior in normalizing the clot structure and in stabilizing hemodiluted clots against mechanical and fibrinolytic degradation.

uPA-mediated plasminogen activation is enhanced by polyphosphate.

Tissue plasminogen activator (tPA) and urokinase (uPA) differ in their modes of action. Efficient tPA-mediated plasminogen activation requires binding to fibrin. In contrast, uPA is fibrin independent and activates plasminogen in solution or when associated with its cellular receptor uPAR. We have previously shown that polyphosphate (polyP), alters fibrin structure and attenuates tPA and plasminogen binding to fibrin, thereby down-regulating fibrinolysis. Here we investigate the impact of polyP on uPA-mediated fibrinolysis. As previously reported polyP of an average chain length of 65 (polyP65) delays tPA-mediated fibrinolysis. The rate of plasmin generation was also delayed and reduced 1.6-fold in polyP65-containing clots (0.74 ± 0.06 vs. 1.17 ± 0.14 pM/s in P < 0.05). Analysis of tPA-mediated fibrinolysis in real-time by confocal microscopy was significantly slower in polyP65-containing clots. In marked contrast, polyP65 augmented the rate of uPA-mediated plasmin generation 4.7-fold (3.96 ± 0.34 vs. 0.84 ± 0.08 pM/s; P < 0.001) and accelerated fibrinolysis (t1/2 64.5 ± 1.7 min vs. 108.2 ± 3.8 min; P < 0.001). Analysis of lysis in real-time confirmed that polyP65 enhanced uPA-mediated fibrinolysis. Varying the plasminogen concentration (0.125 to 1 µM) in clots dose-dependently enhanced uPA-mediated fibrinolysis, while negligible changes were observed on tPA-mediated fibrinolysis. The accelerating effect of polyP65 on uPA-mediated fibrinolysis was overcome by additional plasminogen, while the down-regulation of tPA-mediated lysis and plasmin generation was largely unaffected. PolyP65 exerts opposing effects on tPA- and uPA-mediated fibrinolysis, attenuating the fibrin cofactor function in tPA-mediated plasminogen activation. In contrast, polyP may facilitate the interaction between fibrin-independent uPA and plasminogen thereby accelerating plasmin generation and downstream fibrinolysis.

Factor XIII-A: An Indispensable "Factor" in Haemostasis and Wound Healing.

Factor XIII (FXIII) is a transglutaminase enzyme that catalyses the formation of ε-(γ-glutamyl)lysyl isopeptide bonds into protein substrates. The plasma form, FXIIIA2B2, has an established function in haemostasis, with fibrin being its principal substrate. A deficiency in FXIII manifests as a severe bleeding diathesis emphasising its crucial role in this pathway. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage. The cellular form, a homodimer of the A subunits denoted FXIII-A, was perceived to remain intracellular, due to the lack of a classical signal peptide for its release. It is now apparent that FXIII-A can be externalised from cells, by an as yet unknown mechanism. Thus, three pools of FXIII-A exist within the circulation: plasma where it circulates in complex with the inhibitory FXIII-B subunits, and the cellular form encased within platelets and monocytes/macrophages. The abundance of this transglutaminase in different forms and locations in the vasculature reflect the complex and crucial roles of this enzyme in physiological processes. Herein, we examine the significance of these pools of FXIII-A in different settings and the evidence to date to support their function in haemostasis and wound healing.

Monocytes Expose Factor XIII-A and Stabilize Thrombi against Fibrinolytic Degradation.

Factor XIII (FXIII) is a transglutaminase that promotes thrombus stability by cross-linking fibrin. The cellular form, a homodimer of the A subunits, denoted FXIII-A, lacks a classical signal peptide for its release; however, we have shown that it is exposed on activated platelets. Here we addressed whether monocytes expose intracellular FXIII-A in response to stimuli. Using flow cytometry, we demonstrate that FXIII-A antigen and activity are up-regulated on human monocytes in response to stimulation by IL-4 and IL-10. Higher basal levels of the FXIII-A antigen were noted on the membrane of the monocytic cell line THP-1, but activity was significantly enhanced following stimulation with IL-4 and IL-10. In contrast, treatment with lipopolysaccharide did not upregulate exposure of FXIII-A in THP-1 cells. Quantification of the FXIII-A activity revealed a significant increase in THP-1 cells in total cell lysates following stimulation with IL-4 and IL-10. Following fractionation, the largest pool of FXIII-A was membrane associated. Monocytes were actively incorporated into the fibrin mesh of model thrombi. We found that stimulation of monocytes and THP-1 cells with IL-4 and IL-10 stabilized FXIII-depleted thrombi against fibrinolytic degradation, via a transglutaminase-dependent mechanism. Our data suggest that monocyte-derived FXIII-A externalized in response to stimuli participates in thrombus stabilization.

Fibrinogen Replacement Therapy for Traumatic Coagulopathy: Does the Fibrinogen Source Matter?

Fibrinogen is the first coagulation protein to reach critically low levels during traumatic haemorrhage. There have been no differential effects on clinical outcomes between the two main sources of fibrinogen replacement: cryoprecipitate and fibrinogen concentrate (Fg-C). However, the constituents of these sources are very different. The aim of this study was to determine whether these give rise to any differences in clot stability that may occur during trauma haemorrhage. Fibrinogen deficient plasma (FDP) was spiked with fibrinogen from cryoprecipitate or Fg-C. A panel of coagulation factors, rotational thromboelastography (ROTEM), thrombin generation (TG), clot lysis and confocal microscopy were performed to measure clot strength and stability. Increasing concentrations of fibrinogen from Fg-C or cryoprecipitate added to FDP strongly correlated with Clauss fibrinogen, demonstrating good recovery of fibrinogen (r2 = 0.99). A marked increase in Factor VIII, XIII and α2-antiplasmin was observed in cryoprecipitate (p < 0.05). Increasing concentrations of fibrinogen from both sources were strongly correlated with ROTEM parameters (r2 = 0.78-0.98). Cryoprecipitate therapy improved TG potential, increased fibrinolytic resistance and formed more homogeneous fibrin clots, compared to Fg-C. In summary, our data indicate that cryoprecipitate may be a superior source of fibrinogen to successfully control bleeding in trauma coagulopathy. However, these different products require evaluation in a clinical setting.

Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi.

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s-1) and high shear (1690 s-1) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA-PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.

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 PS-exposing platelets with its cofactor, vitronectin, and fibrinogen. Inclusion of tirofiban or Gly-Pro-Arg-Pro significantly attenuated exposure of PAI-1, indicating a crucial role for integrin αIIbß3 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 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 αIIbß3 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.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70 Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by ßFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not ßFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP.

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy.

Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.

Defective α2 antiplasmin cross-linking and thrombus stability in a case of acquired factor XIII deficiency.

Acquired factor XIII (FXIII) deficiency is a rare and life-threatening condition that is often misdiagnosed or missed completely. A 72-year-old woman presented with symptoms of major unprovoked bleeding but routine coagulation screening tests and platelet count were normal. Low activated FXIII (FXIIIa) activity levels and abnormal urea clot stability led to diagnosis of acquired FXIII deficiency. A modified Bethesda inhibitor titre of 1.6 Bethesda units/ml indicated the presence of a FXIII inhibitor. Bleeding responded to a single dose of FXIII concentrate and immunosuppression with prednisolone induced remission. A subsequent relapse was treated with combined prednisolone and Rituximab resulting in a prolonged, ongoing remission. Here we analyse the mechanisms underlying this idiopathic case of acquired FXIII deficiency. Prospective analysis of patient plasma revealed minimal FXIIIa activity and antigen in presentation and relapse samples. Thrombi formed from these samples lysed rapidly and showed an absence of cross-linked α2 AP. Western blotting revealed the presence of FXIII-B, indicating only FXIII-A and FXIII-A2 B2 were affected. FXIII activity and antigen levels normalised on remission. Our data suggest the presence of inhibitor-induced clearance of FXIII from plasma. As a consequence, reduced thrombus stability was evident due to defective α2 AP cross-linking, thereby explaining symptoms of excessive bleeding.

Platelet-Mediated Modulation of Fibrinolysis.

Platelets are crucial to the hemostatic response. Their role in coagulation is well documented and they have been considered for some time to promote resistance of thrombi to fibrinolysis. Platelets confer resistance to lysis by promoting clot retraction of the immediate fibrin network and through release of plasminogen activator inhibitor-1 from their α-granules. However, recent developments in the field indicate that the role of platelets in fibrinolysis is much more diverse. Indeed, novel studies suggest that platelets form different subpopulations upon activation that play varied roles in regulating hemostasis. Likewise the developments in our understanding of thrombus formation, architecture, and changes in fibrin deposition and composition suggest that these different subpopulations of platelets may populate distinct areas within thrombi and potentially dictate the local hemostatic balance in these areas. This review will discuss the diverse roles of platelets in fibrinolysis and highlight the recent developments in the field and the contribution of both the intracellular pool of modulators as well as the membrane surface in regulating these processes.

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

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 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.