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International Journal of Molecular... Jun 2023CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses...
CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.
Topics: Humans; Blood Platelets; CD40 Ligand; Chemokine CXCL12; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; rho-Associated Kinases; Ristocetin; von Willebrand Factor; rac GTP-Binding Proteins
PubMed: 37298667
DOI: 10.3390/ijms24119716 -
Research and Practice in Thrombosis and... Jul 2023von Willebrand disease (VWD) is considered the most common bleeding disorder and arises from deficiency and/or defect in the adhesive plasma protein von Willebrand... (Review)
Review
von Willebrand disease (VWD) is considered the most common bleeding disorder and arises from deficiency and/or defect in the adhesive plasma protein von Willebrand factor (VWF). Diagnosis of VWD requires clinical assessment and is facilitated by laboratory testing. Several guidelines for VWD diagnosis exist, with the latest American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia 2021 guidelines presenting 11 recommendations, some of which have drawn controversy. In the current narrative review, we provide additional context around difficulties in laboratory diagnosis/exclusion/typing of VWD, with a focus on developing countries/resource-poor settings. In particular, there are many variations in assay methodology, and some methods express high assay variability and poor low-level VWF sensitivity that compromises their utility. Although we favor an initial 4-test assay panel, comprising factor (F) VIII coagulant activity, VWF antigen, VWF glycoprotein Ib binding (VWF:GPIbR or VWF:GPIbM favored over VWF Ristocetin cofactor) and VWF collagen binding, we also provide strategies for laboratories only able to incorporate an initial 3-test assay panel, as favored by the latest guidelines, to improve diagnostic accuracy.
PubMed: 37601016
DOI: 10.1016/j.rpth.2023.102143 -
Platelets Dec 2024Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet...
Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet reactivity and von Willebrand factor (vWF) activity and its mechanism. In this study, we developed a set of microfluidic chips capable of generating different shear gradients and simulated the shear rate distribution in the flow field by COMSOL Multiphysics software. Molecular markers of platelet activation (P-selectin, activated GPIIb/IIIa, phosphatidylserine exposure, and monocyte-platelet aggregate formation) were analyzed by flow cytometry. Platelet aggregation induced by shear gradient was studied by a microfluidic experimental platform, and plasma vWF ristocetin cofactor (vWF: RCO) activity was investigated by flow cytometry. The expression of p-Akt was studied by Western blotting. The results showed that the faster the SGCR, the higher the expression of platelet p-Akt, and the stronger the platelet reactivity and vWF activity. This indicates that fluid shear stress can activate platelets and vWF in a shear gradient-dependent manner through the PI3K/AKT signal pathway, and the faster the SGCR, the more significant the activation effect.
Topics: Humans; von Willebrand Factor; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Platelet Activation; Platelet Aggregation; Blood Platelets
PubMed: 38099316
DOI: 10.1080/09537104.2023.2288679 -
Scientific Reports Dec 2023This in vitro study evaluated the potential hemostatic effect of fresh frozen plasma (FFP) ultrafiltration on clotting factors, coagulation parameters, and plasma...
This in vitro study evaluated the potential hemostatic effect of fresh frozen plasma (FFP) ultrafiltration on clotting factors, coagulation parameters, and plasma properties. ABO-specific units of FFP (n = 40) were prepared for the concentrated FFP and cryoprecipitate. Plasma water was removed from FFP by ultrafiltration using a dialyzer with a pump running at a 300 mL/min. The aliquot of each concentrated FFP after 50, 100, 200, and 250 mL of fluid removal were measured the standard coagulation assay, clotting activity, and plasma properties to compare those parameters of cryoprecipitate. Concentrated FFP contained 36.5% of fibrinogen in FFP with a mean concentration of 7.2 g/L, lower than the cryoprecipitate level. The levels of factor VIII (FVIII), von Willebrand factor (VWF):antigen (Ag), and VWF:ristocetin cofactor (RCo) were also lower in concentrated FFP, whereas the levels of factor V, factor IX, factor XIII, antithrombin and albumin was higher in concentrated FFP. Maximum clot firmness (MCF) in thromboelastometry was approximately one-half of that in cryoprecipitate. Although the levels of VWF:Ag, VWF:RCo, and FVIII differed depending on the ABO blood types, fibrinogen levels, and MCF were not significantly different among the ABO blood groups in FFP and concentrated FFP.
Topics: Hemostatics; von Willebrand Factor; Ultrafiltration; Renal Dialysis; Factor VIII; Fibrinogen; Plasma; Factor V
PubMed: 38062086
DOI: 10.1038/s41598-023-48759-1 -
Journal of Blood Medicine 2023Recombinant von Willebrand factor (rVWF, vonicog alfa, Vonvendi/Veyvondi, Takeda Pharmaceuticals USA, Lexington, MA) and several plasma-derived VWF/factor VIII...
BACKGROUND
Recombinant von Willebrand factor (rVWF, vonicog alfa, Vonvendi/Veyvondi, Takeda Pharmaceuticals USA, Lexington, MA) and several plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrates are available for treating bleeding episodes in patients with von Willebrand disease (VWD).
PURPOSE
To develop population pharmacokinetic (PK)/pharmacodynamic (PD) models that describe VWF:ristocetin cofactor (VWF:RCo) activity and its relationship with FVIII activity (FVIII:C) over time following intravenous administration of either rVWF or a pdVWF/FVIII concentrate (VWF:RCo/FVIII:C 2.4:1) in patients with VWD; to use the final PK/PD models for an in silico comparison of rVWF and pdVWF/FVIII.
METHODS
The population PK model for rVWF was based on data from four clinical studies in which rVWF was administered to adult patients with VWD type 1, 2 or 3 (phase 1: NCT00816660; phase 3: NCT01410227 and NCT02283268) or severe hemophilia A (phase 1: EudraCT 2011-004314-42). The PK and PK/PD models for pdVWF/FVIII were based on data from the phase 1 study (NCT00816660) in patients with type 3 VWD who received either rVWF plus recombinant FVIII (rFVIII, octocog alfa, ADVATE, Takeda Pharmaceuticals USA, Lexington, MA, USA) or pdVWF/FVIII.
RESULTS
There was a marked difference in clearance following rVWF administration compared with pdVWF/FVIII in type 3 VWD, leading to a ~1.75 longer mean residence time (ie, persistence of VWF:RCo activity in the body) and half-life for rVWF versus pdVWF/FVIII. Simulations showed that following repeated administration of rVWF (50 IU/kg), a FVIII:C activity of >40 IU/dL can be maintained for the full 72 h dosing interval.
CONCLUSION
The slower elimination of VWF:RCo following rVWF administration results in a prolonged effect on FVIII turnover compared with pdVWF/FVIII administration.
PubMed: 37332615
DOI: 10.2147/JBM.S395845 -
Research and Practice in Thrombosis and... Mar 2024Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all"...
BACKGROUND
Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all" approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses.
OBJECTIVES
Our aim was to compare well-used platelet reactivity assays.
METHODS
Blood and platelet-rich plasma obtained from the Framingham Heart Study ( = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits.
RESULTS
A strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity ( = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed ( = 0.375; adenosine diphosphate Optimul E vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid-induced aggregation, and explained substantial variance (β = -1.735; = 4.59 × 10; variance proportion = 46.2%) and P2Y antagonists blocking adenosine diphosphate responses (β = -1.612; = 6.75 × 10; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity.
CONCLUSION
Caution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.
PubMed: 38813256
DOI: 10.1016/j.rpth.2024.102406 -
Clinical Case Reports Aug 2023Bernard Soulier Syndrome should be suspected in patients with bleeding disorder symptoms and significant family history, where consanguineous marriages are common....
Bernard Soulier Syndrome should be suspected in patients with bleeding disorder symptoms and significant family history, where consanguineous marriages are common. Diagnosis can be confirmed using a ristocetin test and a peripheral blood smear.
PubMed: 37529138
DOI: 10.1002/ccr3.7767 -
Medicina (Kaunas, Lithuania) Feb 2024: congenital heart disease (CHD), cyanotic and, to a lesser degree, acyanotic, often are accompanied by coagulation abnormalities, impacting substantially morbidity and...
: congenital heart disease (CHD), cyanotic and, to a lesser degree, acyanotic, often are accompanied by coagulation abnormalities, impacting substantially morbidity and mortality. Until now, no consistent hemostatic patterns have been demonstrated in neonates and children with CHD because they represent a variable and heterogenous population. The aim of the present study is to investigate the hemostatic profile, as well as the role of ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type-1 motives), the cleaving protein of von Willebrand factor (VWF) in neonates with CHD and compare them to healthy age-matched controls. : twenty neonates with a mean gestational age of 37.1 ± 2.5 weeks were included in the CHD group, and 18 healthy neonates with a mean gestational age of 38.2 ± 1.5 weeks were in the control group. prothrombin time was significantly prolonged, and accordingly, factor VII (FVII) levels were significantly decreased in the CHD group in comparison to controls. Factor VIII (FVIII), VWF, and ristocetin cofactor activity (Rcof) levels were significantly higher in the study vs. control group. Concentrations of ADAMTS-13 were decreased in the CHD vs. control group, but the difference was not statistically significant. Our results, in combination, indicate a balanced hemostatic mechanism, although with greater variability in neonates with CHD, while developmental aspects of coagulation are evident in the specific patient population. the coagulation profile is moderately impaired early in the course of CHD, though increased thrombogenicity is already present and should not be ignored.
Topics: Infant, Newborn; Child; Humans; Infant; von Willebrand Factor; ADAMTS13 Protein; Pilot Projects; Heart Defects, Congenital; Hemostatics
PubMed: 38399555
DOI: 10.3390/medicina60020268 -
Research and Practice in Thrombosis and... Jan 2024Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand...
von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.
BACKGROUND
Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease.
OBJECTIVES
To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome.
METHODS
VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis.
RESULTS
We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (r = 0.470, < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively.
CONCLUSION
VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.
PubMed: 38268521
DOI: 10.1016/j.rpth.2023.102284 -
Journal of the American Chemical Society Nov 2023Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized...
Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.
Topics: Glycopeptides; Hydrogen; Deuterium; Peptides; Glycoproteins; Polysaccharides
PubMed: 37883679
DOI: 10.1021/jacs.3c04068