Sepsis – it is all about the platelets

Sepsis is accompanied by thrombocytopenia and the severity of the thrombocytopenia is associated with mortality. This thrombocytopenia is characteristic of disseminated intravascular coagulation (DIC), the sepsis-associated coagulopathy. Many of the pathogens, both bacterial and viral, that cause sepsis also directly activate platelets, which suggests that pathogen-induced platelet activation leads to systemic thrombosis and drives the multi-organ failure of DIC. In this paper we review the mechanisms of platelet activation by pathogens and the evidence for a role for anti-platelet agents in the management of sepsis

How not to discover a drug - integrins.

Introduction. Integrins are a family of 24 cell adhesion receptors that play a role in the biggest unmet needs in medicine - cardiovascular disease, immunology and cancer. Their discovery promised huge potential for the pharmaceutical industry. Areas covered. Over 35-years since their discovery, there is little to show for the hundreds of billions of dollars of investment in anti-integrin drug discovery programmes. In this review the author discusses the reasons for the failure of this promising class of drugs and the future for this class of drugs. Expert opinion. Within 10-years, there was a plethora of potent, specific anti-integrin molecules and since their discovery, many of these agents have entered clinical trials. The success in discovering these agents was due to recently discovered monoclonal antibody technology. The integrin-recognition domain Arg-Gly-Asp (RGD) provided the basis for discovering small molecule inhibitors to integrins - both cyclic peptides and peptidomimetics. Most agents failed in the Phase III clinical trials and those agents that did make it to the market were plagued with issues of toxicity and limited efficacy and were soon replaced with non-integrin targeting agents. Their failure was due to a combination of poor pharmacokinetics and pharmacodynamics, complicated by the complex pathophysiology of integrins.</div

Integrins as drug targets: is there a future?

In 1981 fibroblasts were found to adhere to a surface coated in fibronectin and by 1984 the amino acid sequence Arg-Gly-Asp-Ser (RGDS) was identified in a 30-mer fragment of fibronectin as the site essential for supporting adhesion. The RGD sequence was subsequently found to be a common motif with many proteins containing this sequence such as fibronectin, vitronectin and fibrinogen suggesting a family of RGD-containing proteins that were involved in cell adhesion (Humphries et al. 2006).  The initial characterization of the RGD-dependent fibronectin receptor identified a 140kDa protein. AT the same time work on immune cell antigens showed that two antigens, LFA-1 (T-cells) and Mac-1 (macrophages) while expressed on different cells had similar structures. Both were composed of an a and b subunit. While both a-subunits were different both b- subunits were probably identical. The platelet fibronectin receptor was identified as a complex of glycoproteins (GP) IIb and IIIa. The vitronectin receptor was also found to have two subunits and while it bound RGD peptides it did not bind to fibronectin. Thus, there was a picture developing of a family of heterodimeric adhesion proteins that interact with RGDcontaining proteins. While all bound RGD there was evidence that some of the receptors were specific for certain RGD-containing proteins. It was also evident that leucocytes have a family of 3 a/b heterodimeric proteins that share a common b-subunit. Evidence that an anti-GPIIb/IIIa antibody also bound to leucocytes suggested that GPIIb/IIIa may be related to the leucocyte surface markers. This was confirmed in a study which showed that all of the a-subunits came from the same gene family. The cDNA for an 89 kDa subunit of a glycoprotein that mediates cell adhesion was isolated and characterised and this protein was called integrin. By 1987 it had become clear that these three different receptor families – leucocyte surface receptors, platelet GPIIb/IIIa (aIIbb3) and matrix adhesion receptors - were all related. They were grouped together into a super family of distinct but related receptors called Integrins (Hynes 1987). Some but not all of these receptors bound to a family RGD-containing proteins in an RGD-dependent manner.  Initial evidence suggested that a-subunits only interacted with a single b-subunit (only 3 b- subunits were known) and that ligand specificity was conferred by the a-subunit. Thus, integrins were thought to consist of 3 families based around the unique b-subunits. However, there are currently 18 identified a-subunits and 8 b-subunits and it is clear that some a subunits, especially aV are capable of interacting with multiple b-subunits. Thus, integrins can be grouped into sub-families although the precise arrangement is complex (Cox et al. 2010). One approach to categorising integrins is to use a phylogenetic tree where integrin subunits can be grouped according to similarity in their gene sequences (HightWarburton and Parsons 2019).  This discovery of integrins created great opportunity for drug discovery. Integrins were associated with many major diseases with unmet needs. Their presence on leucocytes suggested a role in autoimmune diseases. Their role as cell adhesion molecules suggested a potential role in cancer and their presence on platelets suggested a role in thrombosis and cardiovascular disease. Furthermore the RGD-dependent nature of the interactions provided a hit compound for a small molecule discovery programme and the discovery of inhibitory monoclonal antibodies provided the potential for a biotechnology solution. While monoclonal antibody technology had been around for over 10-years they had yet to be deployed clinically.</p

Targeting SARS-CoV-2-platelet interactions in COVID-19 and vaccine-related thrombosis.

It is clear that COVID-19 is more than a pneumonia and is associated with a coagulopathy and multi-organ failure. While the use of anti-coagulants does reduce the incidence of pulmonary emboli, it does not help with survival. This suggests that the coagulopathy is more likely to be platelet-driven rather than thrombin-driven. There is significant evidence to suggest that SARS-CoV-2 virions directly interact with platelets to trigger activation leading to thrombocytopenia and thrombosis. I propose a model of multiple interactions between SARS-CoV-2 and platelets that has many similarities to that with Staphylococcus aureus and Dengue virus. As platelet activation and thrombosis are major factors in poor prognosis, therapeutics that target the platelet-SARS-CoV-2 interaction have potential in treating COVID-19 and other virus infections

Personalized dual antiplatelet therapy in acute coronary syndromes: striking a balance between bleeding and thrombosis

Purpose of review: Dual antiplatelet therapy (DAPT)-aspirin in conjunction with a P2Y12 inhibitor-is the cornerstone of managing patients with acute coronary syndromes post-revascularization, but the clinical response is highly variable, with potentially devastating consequences. Herein, we review the mechanisms underpinning said variability and explore emerging approaches to normalizing therapeutic benefit. Recent findings: The potent P2Y12 inhibitors, prasugrel and ticagrelor, exhibit minimal inter-individual variability, replacing clopidogrel in DAPT and achieving greater rates of therapeutic response. However, these benefits decline in later phases when bleeding risk begins to supersede that of ischemia. Guided de-escalation of P2Y12 inhibition as well as shortening DAPT duration have emerged as strategies that retain antithrombotic efficacy while reducing bleeding risk. Aspirin is the other component of DAPT but is also used in isolation for secondary prevention of thrombotic disease. In contrast to the P2Y12 inhibitors, genetic influences on aspirin non-response appear to be outweighed by a triad of clinical factors: non-adherence, enteric aspirin use, and inappropriate dosing according to bodyweight and BMI. Multiple de-escalation strategies for DAPT have been shown to mitigate bleeding risk, but it remains unclear which approach is ideal, necessitating head-to-head investigations to determine which exhibits the most favorable cost-to-benefit ratio. However, there is likely a role for more than one approach in clinical practice, depending on patient risk profile. Our approach to aspirin use is also in need of reassessment: strategies to improve adherence, avoidance of enteric aspirin in cardiac patients, and dose adjustment according to bodyweight and/or BMI are all likely to improve rates of therapeutic response. Moreover, platelet function testing may have a role in identifying patients expected to benefit from primary prophylactic aspirin.</p

Early host interactions that drive the dysregulated response in sepsis.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. While many individual cells and systems in the body are involved in driving the excessive and sometimes sustained host response, pathogen engagement with endothelial cells and platelets early in sepsis progression, are believed to be key. Significant progress has been made in establishing key molecular interactions between platelets and pathogens and endothelial cells and pathogens. This review will explore the growing number of compensatory connections between bacteria and viruses with platelets and endothelial cells and how a better understanding of these interactions are informing the field of potential novel ways to treat the dysregulated host response during sepsis

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke

Background: Data are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. Aims: To assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. Methods: This prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. 'Dipyridamole-high on-treatment platelet reactivity (HTPR)' was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. Results: Dipyridamole-HTPR was identified in 71.4-75% of patients on PFA-100 C-ADP, 83.9-86.8% of patients on VerifyNow P2Y12, and 81.5-83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. Discussion: Additional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.</p