Contribution of Contractility to Platelet Biomechanical Functions in Haemostasis and Wound Healing

Re-organization of the actin cytoskeleton accompanies platelet activation and is a prerequisite for the development of platelet contractility.  Defective platelet contractility can give rise to bleeding diathesis and increases the likelihood of thrombus embolization.  Whether and if so  so, how platelet contractility affects aspects of platelet function other than clot retraction is poorly understood. It is also unclear how physical cues of the local microenvironment at vascular injury sites, especially different adhesion proteins and the fibrous topography of the extracellular matrix inform functional responses through integrin-based substrate adhesions. These may contribute to the development of platelet traction forces, adhesion and spreading, but also to granule secretion, pro-coagulant activity, and ECM  remodelling  and thus, have significance for wound healing beyond haemostasis.  By interrogating contractility at multiple stages of thrombus formation using a combination of myosin IIa inhibition, traction force microscopy, super-resolution microscopy, and in vitro assays, our data showed that impaired contractility affects traction force generation, actin bundling, vinculin morphology, GPVI-stimulated platelet aggregation, and single platelet contraction in aggregates formed by GPVI stimulation. Platelet adhesion to vWF under shear, and alpha granule secretion were independent of contractility.  This data indicates that contractility contributes to platelet GPVI  signalling,  but  doesn’t affect  GPIb-mediated platelet adhesion. Further   research   is   required   to   identify the downstream effectors involved in contractility-dependent GPVI signalling.  Per-resolution microscopy complemented by biophysical methods revealed that adhesion protein identity directed platelet force generation and the dimensionality of formed fibronectin matrix.  Platelets adhered to fibronectin developed high traction forces and pulled fibronectin fibrils along their apical membrane, while platelets on laminin developed lower traction forces and pulled fibrils along their basal membrane.  Platelets assembled fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Our findings have potential implications for maintenance of mechanical thrombus integrity during remodeling and vascular repair.  Using fibrinogen scaffolds of different topographies and quantitative microscopy methods, we observed that platelet interactions in the presence of plasma were greater on nanofibrous fibrinogen scaffolds compared to planar fibrinogen scaffolds. Our data show that nanofibers present a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses, which resulted in robust platelet adhesion and spreading, and alpha granule secretion while minimizing pro-coagulant activity. </p

Violin SuperPlots: visualizing replicate heterogeneity in large data sets.

A recent article in MBoC (Goedhart, 2021) presented a web interface for the creation of ‘SuperPlots’. SuperPlots were introduced by Lord and colleagues last year (Lord et al., 2020) to visualise both cell-level variability within replicates as well as the experimental reproducibility between replicates in one single plot. Simple bar charts or boxplots of mean or median values from experimental replicates mask the contribution of underlying cell-to-cell variations in individual experiments, whereas pooling cell-level data across replicates overemphasises statistical differences. The SuperPlot put forward by Lord et al. uses a beeswarm plot to display the cell-level data color-coded according to the individual replicates, and overlays the mean (or median) and error bars (standard deviation or confidence intervals) of each replicate (Figure 1a). The new web interface (Goedhart, 2021) offers an online option for researchers to generate beeswarm SuperPlots, as well as RainCloud plots (Allen et al., 2021), using their own data. We welcome the transparency brought by SuperPlots and would like to introduce an augmentation, the Violin SuperPlot, to further simplify visual inspection of raw data containing large sample sizes

A handshake between platelets and neutrophils might fuel deep vein thrombosis

Deep vein thrombosis (DVT) describes the clogging of veins in extremities, leading to pain, swelling, and potentially life-threatening pulmonary embolism. Although several known factors can provoke DVT, it is not entirely clear how they contribute to thrombus formation. Long-term immobilization of hospitalized patients is thought to provoke DVT through reducing blood flow in the legs. While the associated ischemia activates endothelial cells, two mechanisms drive thrombosis: hypercoagulability caused by the dysregulation of clotting factors, or inflammation caused by the recruitment and activation of immune cells, especially neutrophils. Platelets play an important role in DVT, but how they help to attract neutrophils is incompletely understood. Two independent studies published in the Journal of Thrombosis and Hemostasis and in eLife now provide a fresh perspective on how this might work.</p

Nanofiber topographies enhance platelet-fibrinogen scaffold interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. In this study, we fabricate planar and nanofiber scaffolds from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine (PS) exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy (SEM). Different fibrinogen topographies equally support the spreading and granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. Our data show that the enhanced interactions of platelets with nanofibers results from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets towards robust adhesion formation and α-granule secretion while minimizing their pro-coagulant activity. Similar results on fibrinogen-coated PDMS substrates with micron-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.</p

Successful kidney transplantation normalizes platelet function

Background Uraemic platelet dysfunction is not completely understood, in part due to non-physiological platelet function assays. We have developed a physiological flow-based assay that quantifies platelet function in microlitre volumes of blood under arterial shear. The aim of this study was to characterize platelet function before and after kidney transplantation. Methods Ten patients scheduled for living donor kidney transplant surgery and nine healthy controls were analysed using the assay. The motional parameters of platelet behaviour on von Willebrand factor (VWF) were recorded using customized platelet tracking software. The assay was repeated 3–8 weeks post-transplant in the transplant group and at an interval of >3 weeks in normal healthy volunteers. Results Platelet–VWF interactions were markedly reduced in the 10 pre-transplant patients compared with the healthy controls. In seven patients with immediate graft function, dynamic platelet function returned to normal (despite a small decrease in haemoglobin and haematocrit), but remained markedly abnormal in the three patients with delayed graft function (DGF). Conclusions Dynamic platelet function returned to normal following transplantation in those with immediate graft function. This early improvement was not observed in those with DGF. There may be important clinical implications, as patients with DGF are more likely to undergo invasive procedures, including transplant biopsies and insertion of central venous catheters.</p

Platelets exploit fibrillar adhesions to assemble fibronectin matrix revealing new force-regulated thrombus remodeling mechanisms

Upon vascular injury, platelets are crucial for thrombus formation and contraction, but do they directly initiate early tissue repair processes? Using 3D super-resolution microscopy, micropost traction force microscopy, and specific integrin or myosin IIa inhibitors, we discovered here that platelets form fibrillar adhesions. They assemble fibronectin nanofibrils using αIIbβ3 (CD41/CD61, GPIIb-IIIa) rather than α5β1 integrins, in contrast to fibroblasts. Highly contractile platelets in contact with thrombus proteins (fibronectin, fibrin) pull fibronectin fibrils along their apical membrane, whereas platelets on basement membrane proteins (collagen IV, laminin) are less contractile generating less stretched planar meshworks beneath themselves. As probed by vinculin-decorated talin unfolding, platelets on fibronectin generate similar traction forces in apical fibrillar adhesions as fibroblasts do. These are novel mechanobiology mechanisms by which platelets spearhead the fibrillogenesis of the first de novo ECM, including its 2D versus 3D network architectures depending on their ECM environment, and thereby pave the way for cell infiltration

Corrigendum to Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 1, January 2024, 102322]

The authors regret that the source for Glenzocimab was not acknowledged in the article. Glenzocimab was kindly provided under a material transfer agreement by Acticor Biotech SA. The authors would like to apologise for any inconvenience caused.</p

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.</p

Breast cancer cells mediate endothelial cell activation, promoting von Willebrand Factor release, tumour adhesion and transendothelial migration.

Background: Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis. Objective: To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration. Methods: von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration. Results and conclusion: Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.</p

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease

MYH9-related disease patients with mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in MYH9 -related disease patients, and treatment with tranexamic acid can improve the hemostatic function. Teaser Impaired hemostasis in Myh9 mutant mice due to reduced platelet forces can be improved by tranexamic acid