Introducing a Novel Biophysical Platelet Function Panel to Investigate Disorders of Primary Hemostasis and Bleeding of Unknown Cause

A subset of patients with chronic bleeding remain undiagnosed even after extensive diagnostic evaluation are labeled as "bleeding of unknown cause" (BUC). The key barrier to treating these patients is that they have a clinical bleeding tendency in the presence of normal diagnostic tests, and optimal methods for monitoring and treating patients with BUC remain unknown. While patients with BUC have symptoms of a primary hemostatic disorder, there is no diagnostic test or biomarker that can accurately identify which patients are at risk for bleeding such as those with mild Von Willebrand Disease (VWD) which comprise a broad spectrum of patients with varying degrees of bleeding. In order to fill this diagnostic gap in disorders of primary hemostasis, there is a clinical need for more assays of platelet function. To that end, we have engineered multiple new biophysical assays to assess disorders of primary hemostasis and apply this panel of platelet function testing to potentially define new bleeding disorders, characterize platelet phenotypes in patients with BUC, and refine the definition of mild VWD. Our panel of platelet function tests (Fig 1) collectively enables us to simultaneously assess different facets of primary hemostasis from the microscopic level of single-platelet physiology to hemostatic plug formation, thereby capturing various aspects of platelet function with a single blood sample. Our platelet function panel ranges from platelet adhesion and bulk clot contraction assays to spatially-regulated platelet granule secretion assay, single-platelet contraction cytometry, microfluidics, and a microengineered vascularized bleeding model. As such, we are leveraging these biophysical assays to correlate platelet function with bleeding phenotype severity and establish the dynamic range of this diagnostic panel. We have now established that our assays can be utilized to study blood samples from patients with disorders including hemophilia A, Hermansky-Pudlak Syndrome, FLI-1 mutation, and sickle cell disease among others (Fig 2), demonstrating the clinical utility of our platelet function panel. Our panel can also be used to assess the effects of novel therapeutics on different aspects of platelet function simultaneously. To investigate how crizanlizumab (p-selectin inhibitor) affects hemostatic plug formation, healthy human blood was treated with crizanlizumab. Platelet α-granule secretion enables exposure of P-selectin, and with crizanlizumab we observed restricted platelet filopodial extension and diminished α-granule exocytosis, and an overall decrease in adhered platelets to the fibrinogen micropattern (Fig 3A). The adhesion assay demonstrated a decrease in spreading and adhesion of platelets to collagen and fibrinogen with treatment (Fig 3B). Using the bleeding model, hemostasis was achieved within the normal established range and platelets contracted normally. This suggests that p-selectin has a limited role in the setting of minor injury. Utilizing the bulk contraction assay, we exhibited increased contraction early in clot formation, however over time the treated platelets contracted similarly to the control (Fig 3C). Interestingly, the effect of crizanlizumab-induced restriction of filopodial extension did not correlate with impaired bulk clot contraction or time to form hemostatic plug. Our work suggests crizanlizumab affects platelet spreading at the single-cell level but does not impair platelet function in achieving primary hemostasis at the whole blood level. Here we demonstrate the translational utility of our platelet function panel in providing a deeper understanding of platelet biophysics as it relates to hematologic conditions, with implications for investigation to include pharmaceutical applications. The versatility of this novel panel in capturing platelet function from single-platelet contraction to providing in vitro models with the bleeding device provides multiple dimensions to platelet investigation for primary hemostatic disorders and BUC that have not yet been elucidated. Ongoing research is being conducted using our comprehensive platelet function panel to investigate platelet properties in BUC and mild VWD and correlate these biophysics with bleeding phenotypes. Using this approach we aim to provide novel diagnostic testing with clinical relevance for disorders that have been incompletely characterized until now. Figure 1 Figure 1. Disclosures Meeks: National Institutes of Health: Research Funding; Hemophilia of Georgia: Research Funding; National Hemophilia Foundation: Research Funding; Spark Therapeutics: Consultancy; Sangamo Therapeutics: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy; CSL Behring: Consultancy; Genentech: Consultancy; Takeda: Consultancy. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

The Small Gtpase Rap1 in Platelets Is Critical for Arterial but Not Venous Thrombosis in Mice

Background: Platelets and their main adhesion receptors, integrins, are critical in hemostasis and arterial thrombosis, i.e., in situations involving severe insult to the vasculature and elevated shear stress, respectively. We recently demonstrated that integrin activation under both of these conditions depends on the small GTPase Rap1 directly activating the integrin adapter protein, Talin1. Our studies further suggested that the Rap1-talin1 axis is less important for platelet function at sites of inflammation, i.e., in situations of mild endothelial insult and low shear stress. Aim: To investigate the contribution of the platelet Rap1-Talin1-integrin signaling axis in the pathogenesis of venous thrombosis (VT). Methods: The following mice with documented deficiencies in platelets were subjected to the inferior vena cava (IVC) stenosis VT model: Rap1amKO (Rap1a fl/fl pf4-Cre+), Rap1bmKO (Rap1b fl/fl pf4-Cre+), or both isoforms (Rap1a fl/flRap1b fl/fl pf4-Cre+ [Rap1dKO]), and Talin1mKO (Tln1 fl/flpf4-Cre+). Thrombus weight was determined 48 hours after flow restriction. Clot contraction and tissue plasminogen activator (tPA)-mediated lysis of whole blood clots were studied ex vivo to characterize effects on clot consolidation and stability . Results: Compared to control mice, thrombus weight was markedly reduced in Talin1mKO mice (13.1±2 and 2.2±1.2 mg, p<0.05), but not Rap1dKO or single knockout mice. Ex vivo clot contraction was significantly impaired in whole blood from Talin1mKO (clot weight: control 13.35±3.4 mg vs. 34.63±4.2 mg), Rap1amKO (control 18.0±3.9 mg vs. 25.6±6.0 mg), Rap1bmKO (control 18.0±3.9 vs 23.0±6.6 mg) and in Rap1dKO (control 18.03±3.9 mg vs. 30.02±4.6 mg). Clot weights were not significantly different between Rap1dKO and Talin1mKO mice. However, ex vivo clot lysis assays revealed that compared to controls, clots formed in whole blood from Talin1mKO mice lysed faster, whereas clots from Rap1dKO did not. Conclusions: Platelet Talin1 is critical for VT in mice, and this role is partially independent of its best-known upstream regulator, Rap1. Future studies will identify the alternative mechanism allowing for Rap1-independent Talin1 signaling and platelet function in VT and how these signaling affect various phases of VT. Disclosures Wolberg: Takeda: Research Funding; Bristol Myers Squibb: Research Funding; CSL Behring: Consultancy.

Force-Independent Cleavage of Talin by Calpain Promotes Platelet-Mediated Fibrin Clot Contraction

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino-termini and mass spectrometry to identify proteolytically-cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, fourteen were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a "mechanosensitive clutch" connecting integrins bound to the extracellular matrix to the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of four- and five-helix bundles. The bundles contain 11 vinculin binding sites (VBS), each of which is an α-helix packed into a bundle interior and requiring a structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Thus, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10–50 μM), while at higher Hcy levels (>50 μM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism.

Quantitative and qualitative changes in blood cells associated with COVID-19

Aim. To establish the relationship of hematological disorders with the pathogenesis, course and outcomes of COVID-19. Methods. We examined 235 hospitalized patients with moderate and severe forms of acute COVID-19 receiving anticoagulants and immunosuppressive drugs. We studied the full blood cell counts and morphology along with the platelet function by flow cytometry in comparison with the clinical features and synthesis of inflammatory markers. To assess platelet contractility, blood clot contraction (retraction) kinetics was used in combination with scanning electron microscopy of platelets and blood clots. Results. Hemolytic anemia, neutrophilia and lymphopenia were associated with immature erythrocytes and leukocytes, indicating activation of hematopoiesis. Contraction of blood clots in COVID-19 was impaired, especially in severe and lethal cases, as well as in the presence of comorbidities, including myeloproliferative and coronary heart diseases and acute cerebrovascular disease. In male patients, the changes in clot contraction were more pronounced. Suppression of clot contraction correlated directly with anemia and coagulopathy, including a high D-dimer level, which confirms the pathogenetic significance of blood clot contraction in COVID-19. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with chronic platelet activation and secondary platelet dysfunction. The structure and cellular composition of blood clots depended on the extent of contraction; clots with impaired contraction were porous, had a low content of deformed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin. Conclusion. Blood cells undergoing both quantitative and qualitative changes are involved in the pathogenesis of COVID-19; the suppressed platelet-driven contraction of intravital blood clots may be a part of the prothrombotic mechanisms.

Significant differences in single-platelet biophysics exist across species but attenuate during clot formation

Key Points Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar. Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences.

Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state.

Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke

Background and Purpose: The purpose was to assess quantitatively and qualitatively the composition and structure of cerebral thrombi and correlate them with the signs of intravital clot contraction (retraction), as well as with etiology, severity, duration, and outcomes of acute ischemic stroke. Methods: We quantified high-resolution scanning electron micrographs of 41 cerebral thrombi for their detailed cellular and noncellular composition and analyzed histological images for the overall structure with the emphasis on red blood cell compression, fibrin age, and the signs of inflammation. Results: Cerebral thrombi were quite compact and had extremely low porosity. The prevailing cell type was polyhedral compressed erythrocytes (polyhedrocytes) in the core, and fibrin-platelet aggregates were concentrated at the periphery; both findings are indicative of intravital contraction of the thrombi. The content of polyhedrocytes directly correlated with the stroke severity. The prevalence of fibrin bundles was typical for more severe cases, while the content of fibrin sponge prevailed in cases with a more favorable course. The overall platelet content in cerebral thrombi was surprisingly small, while the higher content of platelet aggregates was a marker of stroke severity. Fibrillar types of fibrin prevailed in atherothrombogenic thrombi. Older fibrin prevailed in thrombi from the patients who received thrombolytics, and younger fibrin dominated in cardioembolic thrombi. Alternating layers of erythrocytes and fibrin mixed with platelets were common for thrombi from the patients with more favorable outcomes. Thrombi with a higher number of leukocytes were associated with fatal cases. Conclusions: Most cerebral thrombi undergo intravital clot contraction (retraction) that may be of underestimated clinical importance. Despite the high variability of the composition and structure of cerebral thrombi, the content of certain types of blood cells and fibrin structures combined with the morphological signs of intravital contraction correlate with the clinical course and outcomes of acute ischemic stroke.