Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Plasma from patients with vaccine-induced immune thrombotic thrombocytopenia displays increased fibrinolytic potential and enhances tissue-type plasminogen activator but not urokinase-mediated plasminogen activation.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were ∼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.

Between a rock and a hard place: resumption of oral anticoagulant therapy after intracranial hemorrhage.

Intracranial hemorrhage (ICH) is the most feared and lethal complication of oral anticoagulant (OAC) therapy. Resumption of OAC after ICH has long posed a challenge for clinicians, complicated by the expanding range of anticoagulant agents available in modern clinical practice, including direct OACs and, more recently, factor XI and XII inhibitors. A review of the current literature found support for resuming OAC in the majority of patients after ICH based on pooled retrospective data showing that resumption is associated with a lower risk of mortality and thromboembolism without a significantly increased risk of recurrent hemorrhage. The optimal time to resume OAC is less clear; however, the available evidence suggests that the composite risk of both recurrent hemorrhage and thromboembolism is likely minimized, somewhere between 4 and 6 weeks, after ICH in most patients. Specific considerations to guide the optimal resumption time in the individual patient include ICH location, mechanism, and anticoagulant class. Patients with mechanical heart valves and intracerebral malignancy represent high-risk groups who require more nuanced decision making. Here, we appraise the literature with the aim of providing a practical guide for clinicians while also discussing priorities for future investigation.

Decoding the role of platelets in tumour metastasis: enigmatic accomplices and intricate targets for anticancer treatments.

The canonical role of platelets as central players in cardiovascular disease by way of their fundamental role in mediating thrombosis and haemostasis is well appreciated. However, there is now a large body of experimental evidence demonstrating that platelets are also pivotal in various physiological and pathophysiological processes other than maintaining haemostasis. Foremost amongst these is the emerging data highlighting the key role of platelets in driving cancer growth, metastasis and modulating the tumour microenvironment. As such, there is significant interest in targeting platelets therapeutically for the treatment of cancer. Therefore, the purpose of this review is to provide an overview of how platelets contribute to the cancer landscape and why platelets present as valuable targets for the development of novel cancer diagnosis tools and therapeutics.

Venous Thromboembolism and Estrogen-Containing Gender-Affirming Hormone Therapy.

Gender-affirming therapy involves the use of hormones to develop the physical characteristics of the identified gender and suppressing endogenous sex hormone production. Venous thromboembolism (VTE) is a known risk of exogenous estrogen therapy, and while evidence of VTE risk among transgender women using modern gender-affirming hormone therapy (GAHT) is still emerging, it is thought to affect up to 5% of transgender women. Historically, GAHT was associated with a high risk of VTE; however, modern preparations are less thrombogenic mainly due to significantly lower doses used as well as different preparations. This review presents the available literature regarding the following four topics: (1) risk of VTE among transgender women receiving estradiol GAHT, (2) how the route of administration of estradiol affects the VTE risk, (3) perioperative management of GAHT, (4) VTE risk among adolescents on GAHT. There is a need for large, longitudinal studies of transgender women using GAHT to further characterize VTE risk and how this is affected by factors such as patient age, duration of GAHT use, tobacco use, body mass index, and comorbidities. Future studies in these areas could inform the development of clinical guidelines to improve the care of transgender people.

A systems immunology study comparing innate and adaptive immune responses in adults to COVID-19 mRNA and adenovirus vectored vaccines.

Identifying the molecular mechanisms that promote optimal immune responses to coronavirus disease 2019 (COVID-19) vaccination is critical for future rational vaccine design. Here, we longitudinally profile innate and adaptive immune responses in 102 adults after the first, second, and third doses of mRNA or adenovirus-vectored COVID-19 vaccines. Using a multi-omics approach, we identify key differences in the immune responses induced by ChAdOx1-S and BNT162b2 that correlate with antigen-specific antibody and T cell responses or vaccine reactogenicity. Unexpectedly, we observe that vaccination with ChAdOx1-S, but not BNT162b2, induces an adenoviral vector-specific memory response after the first dose, which correlates with the expression of proteins with roles in thrombosis with potential implications for thrombosis with thrombocytopenia syndrome (TTS), a rare but serious adverse event linked to adenovirus-vectored vaccines. The COVID-19 Vaccine Immune Responses Study thus represents a major resource that can be used to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.

Improving the rate of inferior vena cava filter retrieval through multidisciplinary engagement.

Background: The placement of retrievable inferior vena cava (IVC) filters occurs commonly, but retrieval rates remain low. Consequently, there is an unmet clinical need to ensure appropriate follow-up and retrieval of these devices. Objectives: To determine the association between an IVC filter surveillance team with filter retrievals or a documented filter plan, time to retrieval, and incidence of filter complications or recurrent venous thromboembolism. Methods: Ambidirectional cohort study evaluating consecutive IVC filter insertions before and after the implementation of a multidisciplinary surveillance team (MDST). We report an odds ratio (OR) with 95% CIs, adjusted by age, sex, weight, and malignancy status. Results: Overall, 453 patients were included, with 272 individuals in the pre-MDST cohort and 181 individuals in the post-MDST cohort. The MDST was associated with a higher composite primary outcome of IVC filter retrieval or a documented filter plan from 79.4% in the pre-MDST cohort to 96.1% in the post-MDST cohort (OR, 6.44; 95% CI, 3.06-15.84). Compared with the pre-MDST cohort, IVC filter retrieval rates were higher in the post-MDST cohort (52.6%-73.5%, respectively; (OR, 2.50; 95% CI, 1.67-3.78). The MDST was associated with a shorter median time-to-filter retrieval (187-150 days, hazard ratio, 1.78; 95% CI, 1.39-2.29), but there was no significant difference when comparing symptomatic or clinically significant IVC filter complications, recurrent venous thromboembolism, or mortality. Conclusion: Our study demonstrates the importance of a structured program to ensure timely IVC filter retrieval and ultimately improve patient care.

C-reactive protein, immunothrombosis and venous thromboembolism.

C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.

Antithrombotic Therapy in Myocardial Infarction: Historic Perils and Current Challenges-A 70-Year Journey.

There have been numerous and intriguing advancements in antithrombotic therapy for myocardial infarction since it was described in the earliest issues of Thrombosis and Haemostasis. In this article, we revisit historical breakthroughs and describe the four most challenging contemporary themes relating to antithrombotic therapy in myocardial infarction. In all four, the challenge is to find the best balance of reducing specific levels of ischaemic risks without increasing bleeding risk. The first is the question of the optimal duration of dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI). This includes discussion of monotherapy after a period of DAPT. The second relates to the role of genotype and phenotype-guided individualisation of antiplatelet therapy. There is emerging evidence for a role of pheno/genotyping in identifying individuals at high risk for recurrent ischaemic events or in guiding the timing of cardiac surgery for patients on DAPT. The third addresses the increasing evidence for dual pathway inhibition, for example, with rivaroxaban in addition to aspirin in patients where high ischaemic and low bleeding risk is demonstrated. Finally the fourth highlights the challenge of the most appropriate combination of antiplatelet and anticoagulation therapy for patients with known atrial fibrillation after PCI. In most individuals, oral P2Y12 inhibitor therapy combined with a direct acting oral anticoagulant appears to be the best strategy based on the available evidence. Overall, the progress in antithrombotic therapy achieved over the last seven decades is remarkable, however, there are important issues to address and progress still to be made.

Platelets as Central Actors in Thrombosis-Reprising an Old Role and Defining a New Character.

Platelets have long been considered simple anucleate cells that rapidly adhere and aggregate at sites of vascular injury. However, recent in vivo experimental data have shed new light on the platelet response to vascular injury. These data have unexpectedly revealed that platelet thrombus formation is a highly dynamic process and yields a platelet thrombus with a distinct hierarchical structure composed of a "core" of highly activated platelets and a "shell" of platelets in a low activation state. This has given rise to the concept that therapeutic targeting of the propagating thrombus shell may hold promise as a means to target thrombosis while sparing hemostasis. While platelets have been historically considered central to arterial thrombosis, they have been traditionally viewed as minor contributors to the formation of venous thrombosis. However, this concept has recently been challenged with the emergence of a large body of evidence highlighting the important proinflammatory function of platelets. The proinflammatory function of platelets is afforded by their ability to induce neutrophil extracellular trap formation, enhance leucocyte recruitment, and secrete granular contents such as high mobility group protein B1 and polyphosphate. These proinflammatory processes trigger coagulation, via the intrinsic pathway, and are central to the formation of venous thrombosis, a condition now appreciated to be a form of sterile inflammation. These data now place platelets at the center stage in orchestrating the thromboinflammatory response underpinning venous thrombosis and have provided new hope that novel platelet-targeted therapeutics may represent a safe and effective approach to prevent venous thrombosis.

Activated platelets in the tumor microenvironment for targeting of antibody-drug conjugates to tumors and metastases.

Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.

Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging.

Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer using three different imaging modalities. These findings warrant further development of this activated platelet specific scFvGPIIb/IIIa, potentially as a universal marker for cancer diagnosis and ultimately for drug delivery in an innovative theranostic approach.

The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function.

PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.

Platelets are not just for clots.

Although the role of platelets as central mediators of hemostasis and thrombosis has been the primary focus of research into platelet biology for more than a century, over the last decade, nonhemostatic functions of platelets have been increasingly defined. As such, a large body of experimental evidence now exists, which places the platelet as a key player in mediating a diverse range of immune, inflammatory, and malignant disease processes. This review outlines the central mechanisms that underpin the nonhemostatic role of platelets and provides a summary of evidence demonstrating a role for platelets in mediating selected inflammatory, immune, and malignant disease processes.

Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbß3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.