Mohammad Al-Tamimi

Validation of paper-based assay for rapid blood typing

We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μL blood sample on a paper surface where grouping antibodies have already been introduced. A thin film chromatograph tank was used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate, resulting in a high optical density of the spot, with no visual trace in the buffer wicking path. Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper substrates had comparable ability to fix agglutinated blood, while a more porous substrate like Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized conditions, a rapid assay for detection of blood groups was developed by spotting blood to antibodies absorbed to paper and eluted with 200 μL of 0.9% NaCl buffer directly by pipetting. RBCs fixation on paper accurately detected blood groups (ABO and RhD) using ascending buffer for 10 min or using a rapid elution step in 100/100 blood samples including 4 weak AB and 4 weak RhD samples. The assay has excellent reproducibility where the same blood group was obtained in 26 samples assessed in 2 different days. Agglutinated blood fixation on porous paper substrate provides a new, simple, and sensitive assay for rapid detection of blood group for point-of-care applications.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)β(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Soluble Glycoprotein VI Is Raised in the Plasma of Patients With Acute Ischemic Stroke

Background and Purpose— Ischemic stroke induced by thrombosis may be triggered by atherosclerotic plaque rupture and collagen-induced platelet activation. Collagen induces glycoprotein VI shedding. Methods— We measured plasma-soluble glycoprotein VI (sGPVI) by enzyme-linked immunosorbent assay in 159 patients with acute (<7-day) ischemic stroke and age/sex-matched community-based control subjects. Results— sGPVI was elevated in stroke compared with controls (P=0.0168). ORs were higher in Quartile 4 for stroke cases (P=0.0121), and sGPVI was significantly elevated in stroke associated with large artery disease across Quartiles 2 to 4 and small artery disease in Quartile 4. sGPVI decreased 3 to 6 months after antiplatelet treatment, consistent with elevated sGPVI due to platelet activation during the thrombotic event. sGPVI correlated with P-selectin (P=0.0007) and was higher in individuals with the GPVIa haplotype (P=0.024). Conclusion— Glycoprotein VI shedding is implicated in the pathology of acute ischemic stroke.

Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa

This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

Measuring soluble platelet glycoprotein VI in human plasma by ELISA.

Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcγRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of ∼1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 ± 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to ∼7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was ∼10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

Compromised ITAM‐based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background: Receptors on platelets that contain immunoreceptor tyrosine‐based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives: We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results: Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen‐related peptide, or to FcγRII‐activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti‐GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa‐blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full‐length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10‐kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL−1 soluble GPVI by ELISA (normal plasma, ∼15 ng mL−1) and IgG purified from patient plasma caused FcγRIIa‐mediated, EDTA‐sensitive cleavage of both GPVI and FcγRIIa on normal platelets. Conclusions: In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.

Focusing on plasma glycoprotein VI

New methods for analysing both platelet and plasma forms of the platelet-specific collagen receptor, glycoprotein VI (GPVI) in experimental models or human clinical samples, and the development of the first therapeutic compounds based on dimeric soluble GPVI-Fc or anti-GPVI antibody-based constructs, coincide with increased understanding of the potential pathophysiological role of GPVI ligand binding and shedding. Platelet GPVI not only mediates platelet activation at the site of vascular injury where collagen is exposed, but is also implicated in the pathogenesis of other diseases, such as atherosclerosis and coagulopathy, rheumatoid arthritis and tumour metastasis. Here, we describe some of the critical mechanisms for generating soluble GPVI from platelets, and future avenues for exploiting this unique platelet-specific receptor for diagnosis and/or disease prevention.

Anti-glycoprotein VI monoclonal antibodies directly aggregate platelets independently of FcgammaRIIa and induce GPVI ectodomain shedding.

Adhesion of circulating platelets to the blood vessel wall initiates thrombus formation in haemostasis and thrombotic disease. The platelet collagen receptor, glycoprotein (GP) VI, is critical for thrombus formation at arterial shear rates and is a potential therapeutic target for anti-thrombotic drugs. In this study, we evaluate eight newly-derived, purified murine anti-human GPVI monoclonal antibodies (mAbs) for their effect on GPVI-dependent platelet aggregation and GPVI ectodomain shedding. All mAbs were raised against the ligand-binding GPVI ectodomain encompassing two immunoglobulin domains (residues 21-234, excluding the signal sequence) and recognized full-length GPVI in human platelet lysates by western blotting. The majority of antibodies induced aggregation in both human platelet-rich plasma (PRP) and washed platelets independently of the Fc receptor, FcγRIIa (not inhibited by the blocking anti-FcγRIIa mAb, IV.3), whereas one mAb (11A7) neither induced aggregation nor inhibited aggregation in response to GPVI ligands, collagen, and collagen-related peptide (CRP). In contrast, Fab fragments of mAb 12A5 strongly blocked collagen- and CRP-, but not convulxin-induced aggregation. In addition, it is shown for the first time in vitro that anti-GPVI mAbs can induce metalloproteinase-dependent ectodomain shedding of human GPVI, generating an ∼10-kDa remnant that remained platelet-associated and an ∼55-kDa soluble fragment. In conclusion, this analysis of anti-GPVI mAbs provides useful tools for studying the functional role of platelet GPVI.

Engineering paper as a substrate for blood typing bio-diagnostics

The effect of paper structure on blood typing visualization was quantified and analyzed to engineer low-cost diagnostics. Commercial and experimental papers varying in fibre composition, basis weight, density and porosity were investigated for their ability to separate agglutinated (blood interacted with specific antibodies) from non-agglutinated (blood interacted with non-specific antibodies) red blood cells (RBCs). Antibodies solutions and blood samples were sequentially absorbed on paper, allowed to interact, eluded with a saline solution, and the intensity of the remaining blood spot was quantified by image analysis. The efficiency and clarity of RBC separation was quantified with the relative intensity (R.I.) index defined as the intensity ratio of the non-specific test over the specific system; the lower the R.I., the better is the separation between a positive from a negative test. Thick and dense papers are improper for blood typing as they retain indiscriminately both agglutinated and non-agglutinated RBCs. Thin and porous papers provided the best performance. The R.I. index (the lower the better) increased fairly linearly with paper density and thickness but inversely proportionally with paper pore size. The type of fibres played a minor role. The paper structure is critical in the design of blood typing assay. However, it is only one element of the diagnostic system to engineer with the interactions RBC-antibody-paper.

The platelet Fc receptor, FcγRIIa

Human platelets express FcγRIIa, the low‐affinity receptor for the constant fragment (Fc) of immunoglobulin (Ig) G that is also found on neutrophils, monocytes, and macrophages. Engagement of this receptor on platelets by immune complexes triggers intracellular signaling events that lead to platelet activation and aggregation. Importantly these events occur in vivo, particularly in response to pathological immune complexes, and engagement of this receptor on platelets has been causally linked to disease pathology. In this review, we will highlight some of the key features of this receptor in the context of the platelet surface, and examine the functions of platelet FcγRIIa in normal hemostasis and in response to injury and infection. This review will also highlight pathological consequences of engagement of this receptor in platelet‐based autoimmune disorders. Finally, we present some new data investigating whether levels of the extracellular ligand‐binding region of platelet glycoprotein VI which is rapidly shed upon engagement of platelet FcγRIIa by autoantibodies, can report on the presence of pathological anti‐heparin/platelet factor 4 immune complexes and thus identify patients with pathological autoantibodies who are at the greatest risk of developing life‐threatening thrombosis in the setting of heparin‐induced thrombocytopenia.