Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Tissue inhibitors of metalloproteinases (TIMPs) modulate platelet ADAM10 activity.

Platelet-specific collagen receptor glycoprotein (GP)VI is stable on the surface of circulating platelets but undergoes ectodomain cleavage on activated platelets. Activation-dependent GPVI metalloproteolysis is primarily mediated by A Disintegrin And Metalloproteinase (ADAM) 10. Regulation of platelet ADAMs activity is not well-defined however Tissue Inhibitors of Metalloproteinases (TIMPs) may play a role. As levels of TIMPs on platelets and the control of ADAMs-mediated shedding by TIMPs has not been evaluated, we quantified the levels of TIMPs on the surface of resting and activated platelets from healthy donors by flow cytometry and multiplex ELISA. Variable levels of all TIMPs could be detected on platelets. Plasma contained significant quantities of TIMP1 and TIMP2, but only trace amounts of TIMP3 and TIMP4. Recombinant TIMP3 strongly ablated resting and activated platelet ADAM10 activity, when monitored using a quenched fluorogenic peptide substrate with ADAM10 specificity. Whilst ADAM10-specific inhibitor GI254023X or ethylenediamine tetraacetic acid (EDTA) could modulate ligand-initiated shedding of GPVI, only recombinant TIMP2 achieved a modest (~20%) inhibition. We conclude that some platelet TIMPs are able to modulate platelet ADAM10 activity but none strongly regulate ligand-dependent shedding of GPVI. Our findings provide new insights into the regulation of platelet receptor sheddase activity.

What do we know? Platelet receptor GPVI initiates platelet adhesion and aggregation and is proteolytically cleaved from the activated platelet surfaceThe metalloproteinases responsible belong to the ADAMs family of enzymes which are inhibited by TIMPsWhat did we discover? Plasma contains significant amounts of TIMP1 and TIMP2Circulating platelets bear significant amounts of TIMPs 1, 2, and 3Recombinant TIMP3 strongly inhibits resting and activated platelet ADAM10 activityExogenous addition of TIMP2 mildly blocked ligand-initiated shedding of GPVIWhat is the impact? TIMPs may modulate ADAM10 activity under resting conditions and stabilize GPVI levels in response to platelet activationAnti-GPVI agents are being evaluated as anti-thrombotic agents, however, acute loss of GPVI in trauma or settings of thrombocytopenia is linked with clinical bleedingUnderstanding how GPVI levels are regulated is important as agents that modulate GPVI function are emerging as important therapeutics for clinical applications in Thrombosis and Hemostasis fields.

Antagonistic Roles of Human Platelet Integrin αIIbß3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear.

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s-1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

Imaging Platelet Processes and Function-Current and Emerging Approaches for Imaging in vitro and in vivo.

Platelets are small anucleate cells that are essential for many biological processes including hemostasis, thrombosis, inflammation, innate immunity, tumor metastasis, and wound healing. Platelets circulate in the blood and in order to perform all of their biological roles, platelets must be able to arrest their movement at an appropriate site and time. Our knowledge of how platelets achieve this has expanded as our ability to visualize and quantify discreet platelet events has improved. Platelets are exquisitely sensitive to changes in blood flow parameters and so the visualization of rapid intricate platelet processes under conditions found in flowing blood provides a substantial challenge to the platelet imaging field. The platelet's size (~2 µm), rapid activation (milliseconds), and unsuitability for genetic manipulation, means that appropriate imaging tools are limited. However, with the application of modern imaging systems to study platelet function, our understanding of molecular events mediating platelet adhesion from a single-cell perspective, to platelet recruitment and activation, leading to thrombus (clot) formation has expanded dramatically. This review will discuss current platelet imaging techniques in vitro and in vivo, describing how the advancements in imaging have helped answer/expand on platelet biology with a particular focus on hemostasis. We will focus on platelet aggregation and thrombus formation, and how platelet imaging has enhanced our understanding of key events, highlighting the knowledge gained through the application of imaging modalities to experimental models in vitro and in vivo. Furthermore, we will review the limitations of current imaging techniques, and questions in thrombosis research that remain to be addressed. Finally, we will speculate how the same imaging advancements might be applied to the imaging of other vascular cell biological functions and visualization of dynamic cell-cell interactions.

Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI.

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.