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Hans Ulrichts

Publications and source records attributed to Hans Ulrichts.

9 recordsLinked to original sources
PubMed2006

Role of glycoprotein Ibalpha in phagocytosis of platelets by macrophages.

BACKGROUND: Platelet (PLT) storage at 0 to 4 degrees C suppresses bacterial multiplication, but induces clusters of glycoprotein (GP) Ibalpha that trigger their phagocytosis by macrophages and reduce their survival after transfusion. A method was sought that detects cold-induced changes in GPIbalpha involved in phagocytosis. STUDY DESIGN AND METHODS: Human PLTs were isolated and stored for up to 48 hours at 0 degrees C. Binding of a phycoerythrin (PE)-labeled antibody directed against amino acids (AA) 1-35 on GPIbalpha (AN51-PE) was compared with phagocytosis of PLTs by matured monocytic THP-1 cells, analyzed by fluorescence-activated cell sorting. RESULTS: Freshly isolated PLTs were detected as a single population of AN51-PE-positive particles and showed less than 5 percent phagocytosis. Cold storage led to a decrease in AN51-PE binding and an increase in phagocytosis. N-Acetylglucosamine, known to interfere with macrophage recognition of GPIbalpha clusters, restored normal AN51-PE binding to cold-stored PLTs and suppressed phagocytosis. CONCLUSIONS: It is concluded that binding of an antibody against AA 1-35 on GPIbalpha reflects changes in GPIbalpha that make PLTs targets for phagocytosis by macrophages.

Acetylglucosamine↗
PubMed2006

Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIba.

We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIb a -von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a cone-and-plate viscometer (shear: 5,000 s(-1) for 5 min) in the presence of antagonists to GPIb a (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared from ACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIb a with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ~ 30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIb a , explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIb a with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.

Abciximab↗
PubMed2005

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V.

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.

Animals↗
PubMed2005

Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1.

OBJECTIVE: In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin. METHODS AND RESULTS: We demonstrate that procoagulant activity is related to a sustained increased [Ca2+]i, which in turn depends on extracellular Ca2+ influx. Increased PtdSer exposure coincides with increased [Ca2+]i and was observed in a subpopulation (approximately 14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbalpha made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca2+]i and procoagulant activity induced by collagen plus thrombin. CONCLUSIONS: The interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced procoagulant activity by inducing a sustained elevation in [Ca2+]i in a subpopulation of platelets.

Apoptosis Regulatory Proteins↗
PubMed2005

How does agkicetin-C bind on platelet glycoprotein Ibalpha and achieve its platelet effects?

The platelet glycoprotein (GP) Ib-IX-V receptor complex has a central role in primary haemostasis and possesses binding sites for the plasmatic adhesive protein von Willebrand Factor (VWF) and thrombin. Several snake venom components have been identified in recent years that target this receptor complex and modulate its functionality. Among them, agkicetin-C is from Deinagkistrodon acutus and proved to be a potent antagonist of GPIb-IX-V. We further studied the structure-activity relationships of agkicetin-C in order to reveal the molecular mechanisms of its antagonistic effect. Agkicetin-C concentration-dependently inhibited botrocetin-, ristocetin- and low dose thrombin- (0.32-0.4nM) induced platelet aggregation. Moreover, it abolished platelet adhesion to collagen under high shear conditions (2600/s), while having only minor effects at low shear rate (650/s), which suggested it targets mainly GPIbalpha instead of other platelet glycoproteins. The interaction site of agkicetin-C was further refined: it recognizes a linear sequence in a recombinant GPIbalpha (AA1-289) fragment and inhibited completely the ristocetin-induced VWF binding to this fragment. Using cross-blocking studies with epitope-mapped anti-GPIbalpha monoclonal antibodies, the binding region of agkicetin-C was refined to the AA201-282 region. In conclusiong the C-type lectin agkicetin-C is a potent GPIb-IX-V antagonist, inhibiting both VWF and thrombin interaction through binding to the AA201-282 region in GPIbalpha. Another thing of interest is that, although agkicetin-C did not agglutinate platelets in all conditions tested in vitro, it caused a severe thrombocytopenia in rats, suggesting a different mechanism than with flavocetin-A or echicetin.

Amino Acid Sequence↗
PubMed2004

Identification of residues within human glycoprotein VI involved in the binding to collagen: evidence for the existence of distinct binding sites.

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

Amino Acid Sequence↗
PubMed2003

von Willebrand factor but not alpha-thrombin binding to platelet glycoprotein Ibalpha is influenced by the HPA-2 polymorphism.

OBJECTIVE: Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex. The N-terminal domain of the GPIbalpha chain contains binding sites for alpha-thrombin and von Willebrand factor (VWF). The human platelet alloantigen (HPA)-2 polymorphism of the GPIbalpha gene is associated with a C/T transition at nucleotide 1018, resulting in a Thr/Met dimorphism at residue 145 of GPIbalpha. To study the structural and functional effects of this dimorphism, N-terminal fragments (AA1-289) of the HPA-2a and HPA-2b alloform of GPIbalpha expressed in CHO cells were used. METHODS AND RESULTS: Of 74 moAbs directed against human GPIbalpha, 2 antibodies with epitope between AA1-59 could differentiate between both alloforms. In addition, VWF bound with a higher affinity to the recombinant HPA-2a fragment or to homozygous HPA-2a platelets. In contrast, no difference was found in the binding of alpha-thrombin to the recombinant alloform fragments or of antibodies directed against the alpha-thrombin binding anionic sulfated tyrosine sequence (AA269-282). CONCLUSIONS: Whereas the Thr145Met dimorphism does not affect alpha-thrombin binding, it does influence the conformation of the N-terminal flanking region and first leucine-rich repeat of GPIbalpha and by this has an effect on VWF binding.

Animals↗
PubMed2003

Integrin alphaIIbbeta3 and shear-dependent action of glycoprotein Ibalpha stimulate platelet-dependent thrombin formation in stirred plasma.

Under conditions of arterial-wall shear rates, platelets bind to von Willebrand factor (vWf) by way of the glycoprotein Ib (GP Ib) complex and integrin alpha(IIb)beta(3). Both adhesive receptors may also play roles in the development of procoagulant activity of platelets. Here, we investigated the effect of shear stress, as provided by a rotating cylinder, on GP Ib- and integrin alpha(IIb)beta(3)-dependent thrombin generation in coagulating platelet-rich plasma (PRP). We measured thrombin continuously with the use of fluorometry from the cleavage rate of a fluorescent low-affinity substrate. The integrin alpha(IIb)beta(3) antagonist abciximab progressively reduced the peak of thrombin formation up to 43% when rate of stirring and shear stress were increased (estimated shear rates of 105-420 s(-1)). Abciximab did not lower the peak of thrombin formation in stirred PRP from patients with Glanzmann's thrombasthenia lacking alpha(IIb)beta(3) but, surprisingly, shortened the time until onset. In PRP from control subjects, antibodies specifically directed against vWf-binding epitopes on GP Ibalpha reduced thrombin formation, with 25% to 30% at the high but not at the low stirring rate. In combination with the anti-GP Ib antibody, abciximab retained its strong inhibitory effect only at the high stirring rate. We conclude that thrombin formation and coagulation in stirred PRP depend, to a large extent, on platelet adhesion to integrin alpha(IIb)beta(3) and, in a shear-dependent way, on GP Ib.

Blood Coagulation↗