Fibrinogenolysis and fibrinolysis in vaccine-induced immune thrombocytopenia and thrombosis.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome associated with adenoviral vector vaccines for COVID-19. The syndrome is characterized by thrombosis, anti-platelet factor 4 (PF4) antibodies, thrombocytopenia, high D-dimer, and hypofibrinogenemia. OBJECTIVES: To investigate abnormalities in fibrinolysis that contribute to the clinical features of VITT. METHODS: Plasma samples from 18 suspected VITT cases were tested for anti-PF4 by ELISA and characterized as meeting criteria for VITT (11/18) or deemed unlikely (7/18; non-VITT). Antigen levels of PAI-1, factor XIII (FXIII), plasmin-α2antiplasmin (PAP), and inflammatory markers were quantified. Plasmin generation was quantified by chromogenic substrate. Western blotting was performed with antibodies to fibrinogen, FXIII-A, and plasminogen. RESULTS: VITT patients 10/11 had scores indicative of overt disseminated intravascular coagulation, while 0/7 non-VITT patients met the criteria. VITT patients had significantly higher levels of inflammatory markers, IL-1ß, IL-6, IL-8, TNFα, and C-reactive protein. In VITT patients, both fibrinogen and FXIII levels were significantly lower, while PAP and tPA-mediated plasmin generation were higher compared to non-VITT patients. Evidence of fibrinogenolysis was observed in 9/11 VITT patients but not in non-VITT patients or healthy controls. Fibrinogen degradation products were apparent, with obvious cleavage of the fibrinogen α-chain. PAP complex was evident in those VITT patients with fibrinogenolysis, but not in non-VITT patients or healthy donors. CONCLUSION: VITT patients show evidence of overt disseminated intravascular coagulation and fibrinogenolysis, mediated by dysregulated plasmin generation, as evidenced by increased PAP and plasmin generation. These observations are consistent with the clinical presentation of both thrombosis and bleeding in VITT.

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.

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.

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.

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 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.

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.

Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network.

Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of 'knots' interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin 'knots'. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin's viscoelastic properties by rheometry. PolyP reduced the stiffness (G') and ability of the fibrin network to deform plastically G'', but to different extents. Consequently, the relative plastic component (loss tangent (G''/G')) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing 'knotted' regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically.

Novel aspects of platelet factor XIII function.

Pools of factor XIII (FXIII) exist in the plasma and within the cytoplasm of hematopoietic cells, including platelets. The functions of the cellular form, FXIII-A, have been assumed to be intracellular in nature, as the protein lacks a signal sequence for its release. Mounting evidence now suggests that platelet FXIII-A modulates hemostasis by several different mechanisms. In this condensed review we discuss recent advances in our understanding of the novel intracellular and extracellular functions of platelet FXIII-A.

Polyphosphate as a haemostatic modulator.

Platelets are small anuclear cells that play a central role in haemostasis. Platelets become activated in response to various stimuli triggering release of their granular contents into the surrounding milieu. One of these types of granules, termed dense granules, have been found to contain polyphosphate (polyP) in addition to other inorganic biomolecules, such as serotonin, ADP, ATP, PPi. Individuals deficient in dense granules exhibit bleeding tendencies, emphasizing their importance in haemostasis. Platelet polyP is of a relatively defined size, approximately 60-100 phosphate monomers in length. These linear polymers act at various points in the coagulation and fibrinolytic systems thereby modulating the haemostatic response. Due to its highly anionic nature, polyP lends itself to being a natural activator of the contact system. The contact system functions in multiple pathways including coagulation, fibrinolysis, inflammation and complement. Activation of the contact system accelerates thrombin generation, the terminal enzyme in the coagulation cascade. PolyP also modulates factors further downstream in the coagulation cascade to augment thrombin generation. The net effect is increased fibrin formation and platelet activation resulting in faster clot formation. PolyP is incorporated into the forming clot thereby modifying the structure of the resulting fibrin network and its susceptibility to degradation by certain plasminogen activators. In conclusion, release of platelet polyP at the site of injury may facilitate clot formation and augment clot stability thereby promoting wound healing.

Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin.

Activated platelets secrete a negatively charged polymer, polyphosphate (polyP). Here, we explore the interactions of polyP with fibrin(ogen) and its effect on fibrin structure and fibrinolysis. Electrophoretic mobility and binding assays indicate that polyP interacts with fibrinogen and soluble fibrin. Clots formed in the presence of polyP exhibited reduced turbidity and permeability indicative of a tighter fibrin network, but these changes were not related to cross-linking or fibrinopeptide release. Microscopy showed a change in fibrin distribution in clots formed with polyP; with formation of tight aggregates of fibrin fibers interspaced with large pores in contrast to homogenous fiber distribution in control clots. Lysis by tissue plasminogen activator (tPA) and plasminogen or plasmin was delayed in clots formed with polyP and depended on both the activator and polyP concentration. Adding polyP to the clot after fibrin formation or to repolymerizing soluble fibrin did not affect lysis, indicating changes induced by polyP occur at the level of conversion of fibrinogen to fibrin. Surface plasmon resonance showed that the presence of polyP reduced the binding of both plasminogen and tPA to partially lysed fibrin surfaces. These data show that polyP directly influences fibrin architecture and attenuates fibrinolysis through reduced binding of fibrinolytic proteins.

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.

Polyphosphate modulates blood coagulation and fibrinolysis.

Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing.