Antiprothrombin antibodies induce platelet activation : a possible explanation for anti‐FXa therapy failure in patients with antiphospholipid syndrome?

Background Arterial and venous thrombosis are both common in antiphospholipid syndrome (APS). Recent studies have shown that anti-factor Xa (FXa) therapy in APS patients leads to a greater number of patients with arterial thrombosis than with warfarin. We hypothesize that this may be due to the lowering of prothrombin levels by warfarin. Objectives To investigate whether antiprothrombin antibodies induce platelet aggregation and to identify the platelet receptors involved. A second aim was to investigate the effect of reduced prothrombin levels on antiprothrombin antibody-induced platelet aggregation. Methods Enzyme-linked immunosorbent assays were performed to measure binding of antiprothrombin antibodies to prothrombin fragment 1+2 and prothrombin. Platelet aggregation assays in washed platelets were performed. Fc gamma RIIA was immunoprecipitated and tyrosine-phosphorylated Fc gamma RIIA was measured by western blot. Results The antiprothrombin antibodies 28F4 and 3B1 had lupus anticoagulant (LAC) activity and caused platelet aggregation in the presence of Ca2+ and prothrombin. Antiprothrombin antibodies without LAC activity did not activate platelets. Inhibition of Syk and Src kinases and Fc gamma RIIA blocked platelet aggregation. Fab and F(ab')(2) fragments of 28F4 were unable to induce platelet aggregation. Immunoprecipitations showed that whole 28F4 immunoglobulin G induced tyrosine phosphorylation of Fc gamma RIIA. Platelet aggregation was significantly reduced when prothrombin levels were reduced from 1 mu M to 0.2 mu M. Conclusions Antiprothrombin antibodies with LAC activity are able to activate platelets via Fc gamma RIIA. Decreased prothrombin levels resulted in less antiprothrombin antibody-mediated platelet aggregation. This may explain the lower incidence of arterial thrombosis in patients treated with warfarin than with anti-FXa therapy

Heparin and heparin proteoglycan-mimetics activate platelets via PEAR1 and PI3Kβ

BACKGROUND: Platelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. OBJECTIVES: We have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. METHODS: A heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. RESULTS: We show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbβ3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kβ, leading to Akt phosphorylation. CONCLUSION: Our findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kβ is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease

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 glycoprotein receptors GPVI, CLEC-2, FcγRIIA and PEAR1 leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signalling cascades. GPVI, CLEC-2 and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase (PI3K). 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 multimerised 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 utilise a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerise the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, ATP secretion and protein phosphorylation were analysed using standardised methods. Results: Multimerisation 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 to the other nanobody-based ligands, despite a sub-nanomolar binding affinity. Conclusions: The multivalent nanobodies represent a series of standardised, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays

Studies of platelet signalling and endothelial cell responses using unique synthetic drugs

Haemostasis is a complex and tightly regulated process which protects us from bleeding. Platelets are essential for maintained haemostasis. Under normal conditions platelets are calmed by antithrombotic substances release by the endothelium. During vascular injury, the platelets will activate and form a haemostatic plug to prevent bleeding. Inflammatory processes like atherosclerosis can disturb the haemostatic balance and lead to severe consequences like myocardial infarction and stroke. Inhibition of platelets and coagulation are common treatments to prevent unwanted blood clot formation. There is a great need for increased knowledge on the mechanisms of thrombosis and characterisation of new substances with possible therapeutic potential. This thesis used unique synthetic drugs to study platelet signalling and endothelial responses. Paper I showed that both sulfated polysaccharides from seaweed and synthetic glycopolymers which mimic their chemical properties caused platelet activation. Paper II elucidated the molecular mechanism underlying platelet activation by sulfated glycopolymers and polysaccharides. We found that human platelet activation took place via the Platelet endothelial aggregation receptor 1 (PEAR1), while mouse platelet activation was mainly via C-type lectin-like receptor 2. Aggregation was supported by Glycoprotein Ibα in both species. Paper III showed the effect of synthetic glycopolymers and natural polysaccharides on cultured human endothelial cells. We found that both the glycopolymers and polysaccharides caused a proinflammatory response after 24h. In Paper IV, the effect of a synthetic purine analogue with a nitrate ester motif was studied. We found that the purine analogue reduced platelet functions by inhibiting Rho-associated protein kinase (ROCK). This thesis describes unique synthetic drugs that can be used for further studies of the mechanisms underlying the biological processes of thrombosis and inflammation. The synthetic glycopolymers can be used to further elucidate the physiological role of PEAR1, a potential future therapeutic target

The Concordance between Immunohistochemical Staining and Silver In Situ Hybridization for HER2 Status in Breast Cancer Tissue Samples

The human epidermal growth factor receptor-2 (HER2) protein has been associated with breast cancer progression and the HER2 status can be used to determine the type of treatment for each breast cancer patient. The purpose of this study was to examine the HER2 protein and gene statuses in breast cancer tissue samples using two methods and analyze the concordance between them. Ten paraffin-embedded, formaldehyde-fixed breast cancer tissue samples from the Biobank at the Department of Pathology and Cytology at Sundsvall Hospital were analyzed in this study. All samples were from women born between 1931 and 1976. The methods used were immunohistochemistry (IHC) to visualise the HER2 protein and silver in situ hybridization (SISH) to detect gene amplification. The IHC staining method is an indirect detection of the HER2 protein using antibodies. The SISH method used in this study is a Dual ISH which detects both the HER2 gene and the centromere region of Chromosome 17 on the same tissue slide. A HER2 gene/Chromosome 17 ratio was calculated according to the manufacturer’s instructions. This ratio was used to determine HER2 gene status. Out of ten samples, seven were positive with IHC and three were negative. The results from the SISH staining exposed a gene amplification in three of the IHC positive samples, while seven samples did not contain any amplified HER2 genes. The conclusion was that the concordance between IHC and SISH for HER2 was 60 percent

Studies of platelet signalling and endothelial cell responses using unique synthetic drugs

Haemostasis is a complex and tightly regulated process which protects us from bleeding. Platelets are essential for maintained haemostasis. Under normal conditions platelets are calmed by antithrombotic substances release by the endothelium. During vascular injury, the platelets will activate and form a haemostatic plug to prevent bleeding. Inflammatory processes like atherosclerosis can disturb the haemostatic balance and lead to severe consequences like myocardial infarction and stroke. Inhibition of platelets and coagulation are common treatments to prevent unwanted blood clot formation. There is a great need for increased knowledge on the mechanisms of thrombosis and characterisation of new substances with possible therapeutic potential. This thesis used unique synthetic drugs to study platelet signalling and endothelial responses. Paper I showed that both sulfated polysaccharides from seaweed and synthetic glycopolymers which mimic their chemical properties caused platelet activation. Paper II elucidated the molecular mechanism underlying platelet activation by sulfated glycopolymers and polysaccharides. We found that human platelet activation took place via the Platelet endothelial aggregation receptor 1 (PEAR1), while mouse platelet activation was mainly via C-type lectin-like receptor 2. Aggregation was supported by Glycoprotein Ibα in both species. Paper III showed the effect of synthetic glycopolymers and natural polysaccharides on cultured human endothelial cells. We found that both the glycopolymers and polysaccharides caused a proinflammatory response after 24h. In Paper IV, the effect of a synthetic purine analogue with a nitrate ester motif was studied. We found that the purine analogue reduced platelet functions by inhibiting Rho-associated protein kinase (ROCK). This thesis describes unique synthetic drugs that can be used for further studies of the mechanisms underlying the biological processes of thrombosis and inflammation. The synthetic glycopolymers can be used to further elucidate the physiological role of PEAR1, a potential future therapeutic target

Is the endogenous ligand for PEAR1 a proteoglycan:clues from the sea

Platelet Endothelial Aggregation Receptor 1 (PEAR1) is an orphan receptor of unknown function which mediates powerful activation of platelets and endothelial cells in response to crosslinking by antibodies and sulfated polysaccharides belonging to the dextran and fucoidan families. PEAR1 is a single transmembrane protein composed of 15 epidermal growth factor-like repeat sequences and with a conserved binding motif, YXXM, which when phosphorylated binds to phosphoinositide 3-kinase (PI3K). The 13th of the repeats has a heparin-binding sequence that is the site of interaction with the sulfated fucoidans and the only known endogenous ligand FcεRIα. Crosslinking of PEAR1 drives Src family kinase phosphorylation of the cytosolic tail leading to binding and activation of PI3K. In this Opinion Article, we summarize the literature on PEAR1 expression, structure and signaling, and the search for further endogenous ligands. We highlight one article in which phosphorylation of a 150 kDa platelet protein by heparin-containing ligands has been reported and propose that PEAR1 is a receptor for one or more glycosaminoglycan-conjugated proteins (proteoglycans). The up-regulation of PEAR1 at sites of inflammation in the vasculature and its role in angiogenesis suggests a role in the interplay of inflammation, platelets, coagulation, and thromboinflammation. We speculate that this may explain the link between single nucleotide variants in PEAR1 and cardiovascular disease