Platelet Fibrinogen Receptor Research Articles

THIS ARTICLE HAS BEEN RETRACTED The impact of a medium molecular weight, low molar substitution hydroxyethyl starch dissolved in a physiologically balanced electrolyte solution on blood coagulation and platelet function in vitro

Background and Objectives Hydroxyethyl starches (HES) may have the potential to impact negatively on haemostasis. Recent findings suggest that side‐effects on haemostasis stem not only from the physicochemical differences between HES, but also from the composition of the solvent. We compared the effects of a newly developed medium molecular weight (MW) and low molar substitution (MS) HES dissolved in a physiologically balanced electrolyte solution (MW 130, MS 0·42; B‐HES) with a commercially available non‐balanced HES (MW 130, MS 0·4; NB‐HES), and with Ringer's lactate (RL) solution in vitro.Materials and Methods Activated partial thromboplastin time (APTT), factor VIII clotting activity (F VIII:C) and von Willebrand factor (vWF) activity were investigated in 48 healthy individuals. Platelet function as measured by turbidimetric platelet aggregometry and whole blood impedance aggregometry induced by adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide (TRAP), and by ADP and TRAP‐induced expression of activated platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa was determined in 24 participants. Haemodilution (25% and 50%, v/v for blood coagulation analyses and 20% and 40%, v/v for platelet function studies) was performed using the two HES preparations and RL.Results APTT was significantly longer and F VIII and vWF significantly lower at 25% and 50% dilutions with NB‐HES compared to B‐HES and RL. At 20% and 40% dilutions, ADP and TRAP‐induced expression of activated platelet surface GP IIb/IIIa was significantly increased by B‐HES compared to NB‐HES and RL. Percentages of platelet GP IIb/IIIa expression were also significantly greater in samples diluted with B‐HES than in undiluted blood. Neither the diluent (B‐HES, NB‐HES and RL) nor the degree of dilution (undiluted, 20% and 40% dilution) had any significant influence on ADP, collagen or TRAP‐induced turbidimetric platelet aggregation or impedance platelet aggregation.Conclusions In contrast to a non‐balanced 130 kDa, MS 0·4 HES (NB‐HES), a 130 kDa, MS 0·42 HES preparation dissolved in a physiologically balanced electrolyte solution (B‐HES) does not affect APTT, F VIII:C and vWF in vitro. Both types of HES do not affect platelet aggregation induced by ADP, collagen or TRAP. B‐HES but not NB‐HES increases the expression of activated platelet GP IIb/IIIa induced by ADP or TRAP.

Integrin β3 genotype influences asthma and allergy phenotypes in the first 6 years of life

The integrin beta3 gene (ITGB3) encodes a subunit of the platelet and monocyte-specific fibrinogen receptor and the widely expressed vitronectin receptor, which have diverse roles in cell migration, adhesion, and signaling. Previous work from our laboratory reported associations between single nucleotide polymorphisms (SNPs) in ITGB3 and asthma and allergic sensitization in 4 populations. To examine whether SNPs in ITGB3 are associated with the development of asthma and allergic phenotypes in early life. We typed 13 SNPs in 206 children participating in a birth cohort study and tested for associations with asthma and allergy phenotypes in the first 6 years of life. Our study revealed significant associations between SNPs in ITGB3 and asthma, wheezing, and IgE levels, suggesting an early role for this gene in the development of asthma and allergy. In particular, SNPs at the 3' end of the gene were significantly associated with IgE levels beginning at 1 year of age, whereas a SNP in intron 1 showed significant interaction effects with viral respiratory illness in infancy on asthma susceptibility. Our results suggest that genetic variation in ITGB3 contributes to asthma susceptibility and allergic sensitization, and that the effects of this gene begin early in life. Similar to our earlier study, different SNPs in the gene are associated with asthma and IgE. ITGB3 may play an important role in the development of asthma and allergy and may represent a potential therapeutic target for the treatment of these disorders.

Smoking and the Platelet Fibrinogen Receptor Glycoprotein IIb/IIIA Pl A1/A2 Polymorphism Interact in the Risk of Lacunar Stroke and Midterm Survival

Smoking, increased fibrinogen levels, and platelet activation are related to the risk of ischemic stroke. The platelet fibrinogen receptor glycoprotein (Gp) IIb/IIIa Pl(A1/A2) polymorphism affects the binding of platelets to fibrinogen and is suggested to interact with smoking. We explored the association of smoking and the Pl(A1/A2) polymorphism with ischemic stroke and survival in the Stroke Aging Memory cohort, comprising 486 consecutive patients (55 to 85 years old) who were analyzed 3 months after an ischemic stroke and followed up for 15 months. Stroke subtype determined by magnetic resonance imaging and GpIIb/IIIa Pl(A1/A2) genotype data were available for 272 patients. In multivariate analysis, smoking was the only factor related to the risk of lacunar infarcts (odds ratio [OR]=1.87, 95% CI=1.05 to 3.31; P=0.033), and it was also a predictor of death (n=24, 8.8%) at 15 months (OR=5.13, 95% CI=1.61 to 16.36; P=0.006), along with age (OR=1.10, 95% CI=1.01 to 1.19; P=0.008). The GpIIb/IIIa Pl(A1/A2) polymorphism alone showed no association with stroke subtype or survival. However, there was a smoking-by-genotype association with the risk of lacunar infarcts (OR=2.10, 95% CI=0.90 to 4.89; P=0.087) and with survival (OR=2.78, 95% CI=0.89 to 8.61; P=0.077). Among younger (55 to 69 years) stroke patients, smokers carrying the Pl(A2) allele were at a higher (OR=5.81, 95% CI=1.26 to 26.80; P=0.024) risk of lacunar infarcts than noncarrier smokers (OR=3.12, 95% CI=1.06 to 9.24; P=0.039). The effect of Pl(A2) and smoking combined on survival was also stronger (OR=8.86, 95% CI=1.68 to 46.55; P=0.010) than the effect of smoking alone (OR=5.06, 95% CI=1.20 to 21.35; P=0.027). Our results indicate that prothrombotic genetic factors may interact with smoking by modifying the stroke phenotype and affecting midterm survival.

CIB1 Positively Regulates Outside-In Signaling through αIIbβ3 by Enhancing FAK and Src Activity.

Integrin αIIbβ3, the platelet fibrinogen receptor, mediates outside-in signaling upon fibrinogen binding. Calcium- and integrin-binding protein 1 (CIB1) specifically interacts with the cytoplasmic domain of αIIb and is involved in platelet spreading. Here we show that CIB1 binding to αIIb is an essential step in the propagation of outside-in signaling through integrin αIIbβ3. Incorporation of BAPTA-AM completely blocked outside-in signaling and CIB-1 binding to aIIb, suggesting that an intracellular Ca2+ rise induced by fibrinogen binding is required for CIB1 interaction with αIIb. When CIB1-binding to αIIb was inhibited by introduction of a function blocking antibody or a synthetic peptide corresponding to αIIb tail, CIB1 localization at the tip of the filopodia, but filopodia formation was not blocked. This result suggests that interaction of CIB1 with αIIb is not required for filopodia formation, but is needed for CIB1 accumulation at the filopodia, as well as platelet spreading. Immunoprecipitation experiments showed that during outside-in signaling, CIB1 associates with FAK. Although association of FAK and CIB1 does not require dynamic rearrangement of the cytoskeleton, their accumulation at the filopodia and the activation of FAK is dependent on cytoskeletal rearrangement, as treatment with cytochalasin D after the platelets form filopodia affects CIB1 localization. Interestingly, the interaction of CIB1 with αIIb upon fibrinogen binding is also necessary for FAK and Src activation. However, Src activity is not required for CIB1 accumulation at the filopodia since this accumulation was not stopped by the Src inhibitor PP2, despite blocking platelet spreading. Our results suggest that during outside-in signaling an intracellular Ca2+ rise occurs that facilitates CIB1 interaction with αIIb. CIB1 recruits FAK to this complex and is localized to the filopodia dependent upon dynamic cytoskeletal rearrangement. Furthermore, activation of Src and FAK requires interaction of CIB1 with αIIb. Although Src activity is not required for the accumulation of CIB1 at the filopodia, it is required for platelet spreading on immobilized fibrinogen. Thus we provide evidence for initial sequence of outside-in signaling in platelets.

The αIIbP258S, a Newly Identified Mutation, and the αIIbG349D Substitutions Cause Glanzmann Thrombasthenia by Impairing αIIb-β3 Association in the Endoplasmic Reticulum.

Glanzmann Thrombasthenia is a recessively inherited hemorrhagic disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3, platelet fibrinogen receptor. We previously analyzed the phenotypic effects of the αIIbG236E substitution, which is located in a strategic point of the highly organized αIIb propeller. We demonstrated that the effect of αIIbG236E is to hamper the association with β3, impairing the correct assembly of the complex. In the present study we analyze the phenotypic effects of two other mutations in the same domain of αIIb. The first mutation, the αIIbP258S substitution, is a newly found missense substitution that was identified during a genetic screening of a cohort of patients with Glanzmann Thrombasthenia coming from Southern Iran. The presence of the mutation was confirmed in several family members affected by Glanzmann Thrombasthenia; no other additional mutations were identified. The effect of the substitution is to eliminate the key proline from blade 4 of αIIb propeller. The second mutation, the αIIbG349D substitution, has been previously described in an Italian patients with Glanzmann Thrombasthenia. It causes a charge substitution in strand D of blade 5 of the propeller. Both substitutions were inserted by site directed mutagenesis in pCDNA3.1 containing normal αIIb cDNA. Human Embryonic Kidney (HEK) cells were then transfected by electroporation with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. After 48 hours cells were analyzed by flow cytometry: the binding of 10E5-FITC (murine anti-human αIIbβ3) in cells expressing αIIbP258Sβ3 or αIIbG349Dβ3 was approximately at the same level of negative controls (cells transfected with αIIb only) and greatly reduced in comparison to the binding to cells transfected with normal αIIbβ3. HEK cells transfected with either αIIbβ3 or αIIbP258Sβ3 or αIIbG349Dβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. All the lysates were reacting at the same level with antibodies directed against β3. When immunoblotting was done in non-reducing conditions utilizing an antibody reacting with αIIb (mab1990) a band corresponding to αIIb was present in both lysates, although less intense in cells transfected with mutants αIIb. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbP258Sβ3 or αIIbG349Dβ3 the ratio pro-αIIb: αIIb was 1:1. In conclusion we report the identification of a new mutation responsible for Glanzmann Thrombasthenia located in αIIb propeller. This mutation, in addition with αIIbG349D, causes Glanzmann Thrombasthenia interfering with αIIbβ3 complex formation in the endoplasmic reticulum as shown by the increase amount of pro-αIIb found in cells expressing these mutants.

Inhibition of Polo-Like Kinase 3 by CIB1 Facilitates Outside-In Signaling through αIIbβ3 in Platelets.

Integrin αIIbβ3, the platelet fibrinogen receptor, is involved in bi-directional signaling during platelet activation. It has been shown that the β3 subunit of this receptor is phosphorylated on both threonine and tyrosine. Although, the role of tyrosine phosphorylation is well studied, the role of threonine phosphorylation is not well understood. Here we identify the kinase responsible for threonine phosphoruylation of β3 and provide a mechanistic significance of this phosphorylation in outside-in signaling. We found that calcium- and integrin-binding protein 1 (CIB1), which specifically interacts with the cytoplasmic domain of αIIb, is not involved in inside-out signaling, but regulates outside-in signaling through αIIbβ3. CIB1 is known to interact with and regulate several serine/threonine protein kinases. Among these, p21 activated kinase 1 (PAK-1) is highly expressed in platelets. We found that CIB1 does not colocalize with PAK-1 during outside in signaling induced by adhesion of platelets to immobilized fibrinogen. When searched for the expression of other kinases that are known to interact with CIB1, we found that polo-like kinase 3 (Plk3) is expressed in platelets and is responsible for the constitutive phosphorylation of β3. Plk3 is constitutively active in resting platelets but is rendered inactive during outside in signaling as a result of CIB1 binding in a calcium dependent manner. Plk3 becomes colocalized with CIB1 at the filopodia when platelets change shape in response to an agonist and remain associated with both CIB1 and αIIb as platelets spread on immobilized fibrinogen, as indicated by immunoprecipitation experiments. These results suggest that in resting platelets, constitutively active Plk3 maintains β3 in a threonine phosphorylated state which is nonpermissive for activation. Upon platelet activation by agonist CIB1 associates with Plk3 and renders it inactive in a calcium-dependent manner. This allows serine/threonine phosphatases, such as PP1-c to dephosphorylate β3 on threonine and facilitate outside-in signaling. Thus we have identified the kinase responsible for threonine phosphorylation of β3 and have provided a possible mechanism of the regulation of outside-in signaling.

Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin αIIb cytoplasmic domain

Integrin αIIb/β3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the αIIb/β3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of αIIb. Using αIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin α2 tail, β3 tail and lamin, but showed a weak binding to the αV tail which shares the highest homology with αIIb tail among the integrin α family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and αIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to αIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with αIIb and may play a critical role in αIIb/β3-mediated platelet function.

Genetic variation in platelet integrin αIIbβ3 (GPIIb/IIIa) and the metastatic potential of renal cell carcinoma

To explore whether genetic polymorphisms in platelet receptors known to be associated with platelet activity have any association with haematogenous metastases in renal cell carcinoma (RCC), as platelets and their fibrinogen receptors may be central to haematogenous cancer spread, in addition to various adhesive proteins on both platelets and tumour cells on themselves. The glycoprotein alpha(IIb)beta3 (GPIIb/IIIa) is the main fibrinogen receptor on platelets, with GPIb-IX-V and GPIa/IIa being the von Willebrand Factor and collagen receptors, respectively. In a cross-sectional survey of 128 men treated for RCC (55 with disseminated disease and 73 with a local disease), they were genotyped using DNA extracted from freshly drawn blood, and central clinical data were recorded. The frequency of possessing the GPIIIaPl(A2) allele among patients with metastatic RCC was 43.6%, and with local disease was 24.7% (P = 0.024). The frequencies of the different alleles of the GIB-IX-V, C3550T and GPIa/IIa C807T polymorphisms did not differ between the groups. In a stepwise logistic regression (metastatic vs local RCC) the Pl(A2) allele remained a significant risk factor, with an odds ratio of 2.7 (95% confidence interval, 1.1-6.5; P = 0.02). The prothrombotic Pl(A2) allele of platelet fibrinogen receptor GPIIb/IIIa increased the risk of metastases in RCC in men.

The Antiplatelet Drug Target in Atherosclerotic Diseases

The aim of this review is (1) to give a rationale for anti-platelet therapy based on mechanisms of platelet rich arterial thrombosis, (2) to point out the pitfalls involved in monitoring therapy with platelet function tests and (3) to outline the potential clinical applications of such therapy based on the various modes of action of anti-platelet drugs. The primary event in arterial thrombosis is platelet-mediated, either due to increased shear or exposed collagen, followed by fibrin-rich thrombosis. Anti-platelet therapy needs to be monitored but most platelet function tests, now in use, do not reflect in vivo function; the anticoagulant used for blood samples removes extra-cellular calcium ions, platelets are often separated before the test, or very high doses of agonist are used: all of these can give misleading results. We review means whereby platelet function can be monitored in whole blood samples anticoagulated with the pure thrombin inhibitor, hirudin. We review the available methods of modifying platelet activity and are particularly interested in agents that do not cause bleeding. Present therapy causes bleeding by interference with COX1, the P2Y(12) receptor or the platelet fibrinogen receptor complex, all of which can be associated with bleeding complications. In contrast, serotonin does not influence formation of haemostatic layers although it is implicated in shear-induced aggregation and thrombus propagation by positive feedback from the large amount of intraplatelet serotonin. We suggest that further investigation of selective serotonin 5HT(2) antagonism would allow effective management of intravascular thrombosis without bleeding complications. This would be safer both as prophylaxis and would also allow cardioprotection of vascular patients undergoing surgical operations.

The formation of platelet–leukocyte aggregates varies during the menstrual cycle

Platelet–leukocyte aggregates are considered to play a significant role in blood coagulation and inflammatory processes. We hypothesized that hormonal changes during the menstrual cycle affect the formation of heterotypic aggregates and therefore may constitute cycle-dependent variations of the susceptibility for thromboembolic events and inflammatory disease. We therefore measured platelet–leukocyte interaction by the determination of platelet–leukocyte aggregates (PLA), platelet P-Selectin expression, and platelet fibrinogen receptor activation by PAC-1 binding in 20 healthy women during their menstrual cycle by flow cytometry. The number of platelet–granulocyte aggregates (PGA) and platelet–monocyte aggregates (PMA) was higher at ovulation compared to any other time-point of the menstrual cycle (p = 0.005, p = 0.022, respectively). Likewise, P-Selectin expression peaked on day 14 (p = 0.040). The course of PLA formation during the menstrual cycle followed the course of estrogen levels, strongly suggesting direct effects of estrogen on platelet–leukocyte interaction. The susceptibility to form platelet–leukocyte aggregates that are inducible in vitro by a suboptimal concentration of thrombin receptor activating peptide-6 decreased slightly during the transition from day 1 to 14 (p = 0.040). These data indicate that platelet function varies during particular phases of the normal menstrual cycle.

Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function.

The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling). Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation. Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.

Recombinant Mini-β3 Integrin Fragments for the Detection of HPA-1 Antibodies.

The αIIbβ3 integrin (GPIIbIIIa, CD61) is the platelet receptor for fibrinogen, fibronectin and vitronectin. A single nucleotide polymorphism (dbSNP Id= rs5918) in the ITGB3 gene defines the Human Platelet Antigen (HPA)-1 system encoding either a Leucine (1a) or Proline (1b) at position 33. HPA-1a immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post transfusion purpura (PTP) and platelet refractoriness. Currently, the detection of HPA-1 antibodies is based on a cumbersome sandwich ELISA requiring HPA genotyped platelets as source of antigen. We set out to express soluble calmodulin (CaM) tagged β3 fragments suitable for HPA-1 antibody detection. The design of fragments was informed by domain structural information, analysis of homology between β3 and integrin-like monomeric proteins and the evolutionary β3 domain phylogeny. Combined with computer modelling, this was the basis of the successful expression in Drosophila S2 cells of eight β3 fragments with either Leu33 or Pro33 (ΔSDL, ΔβA, PSI-Hybrid and PSI alone, both alleloforms for each of the four). Murine monoclonal antibodies Y2/51, SZ21 and CRC54 reacted with the β3 fragments as expected with only CRC54 reacting with the PSI fragment. Two of 3 human HPA-1a specific phage antibodies (19–7, 23–15, both derived from a PTP patient) reacted with all 4 Leu33 fragments but CamTran007 derived from a NAIT patient only reacted with the ΔSDL-Leu33 and ΔβA-Leu33 fragments, defining an epitope split. By ELISA, reactivity of both potency (NIBSC-03/152) and sensitivity (NIBSC-93/710) standards were comparable to those seen by MAIPA. Twelve polyclonal samples from NAIT patients (5 with cerebral haemorrhage [CH], 5 with platelet counts <40x109/L but no CH and 2 with counts >40x109/L) of potency ranging from 5–193 au/ml were detected by ELISA with Leu33 forms of ΔSDL and ΔβA. In conclusion soluble mini-β3-Leu33 fragments have been successfully expressed and shown to react with all polyclonal anti-HPA-1a samples tested from NAIT patients. The CaM tagged β3 fragments are now being further validated with a repository of 150 HPA-1a antibody samples from a NAIT case series with well defined treatment and clinical outcome histories to establish the relation between HPA-1a antibody potency, epitope reactivity and clinical phenotype.

Megakaryocytes Derived from Human Embryonic Stem Cells: A Genetically Tractable System To Study Megakaryocytopoiesis and Integrin Function

The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits αIIb and β3, is required for platelet aggregation, spreading and hemostasis. Platelet agonists such as thrombin and ADP lead to activation of αIIbβ3, thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to αIIbβ3 triggers cytoskeletal changes and granule release (outside-in signaling). Genetic approaches to characterize the molecular pathways involved in αIIbβ3 signaling are not possible with anucleate blood platelets. Therefore we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). αIIbβ3 activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, αIIb+/GPIbα+ cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of αIIbβ3 outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of αIIbβ3 activation. Thus, using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and αIIbβ3 signaling in the native cellular environment.

Activation of Platelet α Iibβ 3 by Exogenous Peptides Corresponding to the Transmembrane Domains of α Iib and β 3.

The platelet fibrinogen receptor α IIbβ 3 exists in an equilibrium between inactive and active conformations. In its inactive conformation, the transmembrane (TM) domains of α IIb and β 3 interact, but they separate when α IIbβ 3 assumes its active conformation. Peptides corresponding to the α IIb TM domain form homodimers in vitro and in bacterial membranes and the interface that mediates this interaction overlaps with the interface that mediates the heteromeric association of the α IIb TM domain with that of β 3. Because the homomeric association of α IIb TM domain is relatively strong, we expected that peptides spanning the α IIb TM domain might activate α IIbβ 3 by binding to the α IIb TM domain, thereby disrupting the α IIb/β 3 TM domain heterodimer. We synthesized a 26 residue peptide corresponding to the α IIb TM domain, flanked by pairs of lysine residues to increase its solubility in water, and assessed its ability to activate washed or gel-filtered platelets in an aggregometer. Addition of 3 μM peptide induced platelet aggregation after a short lag, but unlike aggregation stimulated by ADP, peptide-induced aggregation was not preceded by platelet shape change. Nonetheless, like ADP-induced aggregation, peptide-induced aggregation was inhibited by EDTA and the α IIbβ 3-specific monoclonal antibody A2A9. On the other hand, pre-incubating platelets with PGE1 or apyrase caused only a small decrease in the rate and extent of peptide-induced aggregation, suggesting that the peptide induced aggregation by binding directly to α IIbβ 3. A peptide corresponding the β 3 TM domain behaved in an identical manner, whereas an unrelated peptide, the model TM domain MS-1, had no effect. Similarly, there was little response to an α IIb TM peptide in which glycines 972 and 976, the first and last residues of a GxxxG motif, were changed to leucine. Confirming that the α IIb TM peptide binds to α IIbβ 3, we found that a FITC-labeled α IIb TM peptide co-eluted with purified α IIbβ 3 during size exclusion gel filtration. To determine whether the latter interaction involved the α IIb TM domain, we used the TOXCAT assay. In TOXCAT, the α IIb TM domain-mediated dimerization of a fusion protein containing the α IIb TM domain interposed between maltose binding protein and the ToxR' transcriptional activation domain in the inner membrane of E. coli drives the activation of the chloramphenicol acetyl transferase (CAT) gene. We found that adding the α IIb TM peptide to the bacterial growth medium consistently decreased CAT synthesis, implying that its presence impaired α IIb-mediated fusion protein dimerization. These results provide evidence for the presence of an α IIb/β 3 TM domain heterodimer in unstimulated human platelets and suggest that the GxxxG motif in the α IIb TM domain is involved in its formation. Because the homomeric interaction of α IIb and β 3 TM domains is substantially stronger than their heteromeric interaction, our data are also consistent with the hypothesis that the TM peptides disrupt the heterodimer which functions to maintain α IIbβ 3 in an inactive state. Lastly, the data suggest that stabilizing the α IIb/β 3 TM heterodimer may represent a new approach for the development of novel anti-platelet agents.

Design and Synthesis of Novel Platelet Fibrinogen Receptor Antagonists with 2H‐1,4‐Benzoxazine‐3(4H)‐one Scaffold. A Systematic Study.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The effect of green laser light irradiation on whole blood platelets

Background Laser light irradiation is assumed to have biostimulating effect in various cell types. However, there is still a lack of information concerning response of blood platelets to laser light irradiation. Methods In our study we used flow cytometry to monitor the effect of a green Nd-YAG laser (532 nm, 30 mW) irradiation on platelet activation and the expression of activated GPIIbIIIa glycoprotein complex (fibrinogen receptor) of whole blood platelets stained with fluorolabelled monoclonal antibody PAC-1. Also the formation of platelet microparticles and aggregates in a population of whole blood platelets following such irradiation was evaluated. Results Effects of laser light on platelet activation and reactivity were significant over a wide range of applied energies ( p < 0.01). While low and medium laser light energies (18 and 54 J) increased platelet activation, the irradiation with a high-energy laser light (108 J) resulted in depressed platelet reactivity and attenuated platelet response to activators. In addition, laser light irradiation had significant influence on the formation of platelet microparticles in either resting ( p < 0.05) or ADP-activated ( p < 0.05) platelets, while no significant effect was observed in collagen-activated platelets. On the other hand, laser light irradiation significantly increased the formation of platelet aggregates both in resting ( p < 0.01) and agonists-activated ( p < 0.05) platelets. Conclusions Our results clearly point that the laser light irradiation of blood platelets can trigger signal transduction, leading to platelet activation, as well as the gradual loss of natural platelet reactivity and platelets’ ability to respond to activating agents.

Glycoprotein IIb/IIIa inhibition reduces prothrombotic events under conditions of deep hypothermic circulatory arrest

Deep Hypothermic Circulatory Arrest (DHCA) is employed during thoracic aortic and congenital heart surgery, and can induce postoperative neurological damage probably caused by microthrombembolism. Hypothermia has been reported to induce platelet activation and aggregation. The platelet activation marker P-selectin mediates binding of platelets to leukocytes. Tirofiban and eptifibatide, short-acting inhibitors of the platelet fibrinogen receptor GP IIb/IIIa, have recently been shown to protect platelet function without increasing bleeding during heart surgery using cardiopulmonary bypass. The aim of this study was to investigate the effect of tirofiban and eptifibatide on platelets and platelet-leukocyte interaction under DHCA conditions in vitro. Platelet aggregation, binding of the GP IIb/IIIa activation specific antibody PAC-1, P-selectin expression as well as monocyte and granulocyte content of aggregates were investigated in unstimulated and ADP-stimulated samples using flow cytometry. Tirofiban and eptifibatide inhibited massive platelet aggregation and PAC-1 binding which were induced by DHCA conditions. P-selectin expression was inhibited by tirofiban but increased by eptifibatide at hypothermia. Platelet-bound leukocytes were present in all samples. Eptifibatide increased granulocyte content of aggregates at hypothermia in ADP-stimulated samples. We conclude that under conditions of DHCA both tirofiban and eptifibatide inhibit platelet aggregation but have different effects on platelet P-selectin expression and platelet-leukocyte interaction. Application of a short-acting and non-activating GP IIb/IIIa inhibitor should be considered during DHCA in vivo to prevent occlusion of the microvasculature and subsequent organ damage.

Design and synthesis of novel platelet fibrinogen receptor antagonists with 2 H-1,4-benzoxazine-3(4 H)-one scaffold. A systematic study

New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin α IIbβ 3) antagonists were prepared on a 2 H-1,4-benzoxazine-3(4 H)-one scaffold. Their anti-aggregatory activities in human platelet rich plasma and their affinity towards α IIbβ 3 and α Vβ 3 integrins were assessed. Various substitution positions and side chain variations were studied. In contrast to the generally accepted model, compounds containing ethyl esters as aspartate mimetics were in general more active than the corresponding free acids. We suggest an explanation for the observed behaviour of these new compounds.

Importance of GPVI in Platelet Activation and Thrombus Formation In Vivo.

Collagen is one of the major components of the vessel wall responsible for platelet adhesion and activation at sites of vascular injury. In vitro studies have shown that α2β1 and GPVI directly and αIIbβ3 and GPIb-IX-V indirectly, via vWF, are all involved in the adhesion of platelets to collagen. However, the importance of GPVI on the adhesion and activation of platelets in vivo is still controversial. Here we show that in vivo GPVI plays an important role in platelet adhesion and activation when collagen is exposed to blood. To determine the role of GPVI on thrombus formation, we compared thrombus formation in FcRγ null mice, which do not express GPVI on the platelet surface, and in wild type mice after vessel injury induced by ferric chloride or by a nitrogen dye laser. We studied arterial thrombus formation in the microcirculation of the cremaster and the mesentery muscles using high speed multi channel intravital fluorescence widefield microscopy. Real time platelet accumulation in the developing thrombus was detected using a fluorescent antibody directed against αIIb. After an injury induced by the ferric chloride, we observed a significant delay in both the time to formation of an initial thrombus and the time to vessel occlusion in FcRγ null mice in comparison with wild type mice. When activated platelets isolated from FcRγ null mice were injected into a recipient FcRγ null mouse, we were able to restore the formation of a thrombus. This effect was abolished by injection of Lamifiban, an inhibitor of activated αIIbβ3. These results indicate that GPVI is not only involved in vivo in the adhesion of platelets to collagen but also plays an important role in the activation of the platelet fibrinogen receptor αIIbβ3. In contrast, platelet accumulation after laser-induced injury in the FcRγ null and the wild type mice was comparable. No difference in the kinetics of platelet accumulation into the laser induced growing thrombus was observed. To understand the different pathways leading to thrombus formation in vivo after ferric chloride or laser induced injury, we examined collagen exposure after vessel injury and the accumulation of tissue factor (TF) in the developing arterial thrombus of FcRγ null and wild type mice using antibodies directed against mouse collagen type I and mouse TF. We observed a significant exposure of collagen at sites of thrombus formation after ferric chloride treatment. In contrast, we did not observe any collagen exposure on blood vessels after laser-induced injury. Furthermore, the ratio of TF/platelets present into the thrombus after injury was 5 fold greater after laser injury than after ferric chloride treatment. These results suggest that TF but not collagen plays an important role in thrombus formation induced by laser injury of the vessel wall. Altogether, our results indicate that the GPVI receptor is involved in vivo in platelet adhesion when collagen is exposed to blood and plays an important role in the activation of other platelet integrins such as αIIbβ3 leading to the formation of a stable thrombus.

The procoagulatory effects of delta-9-tetrahydrocannabinol in human platelets.

Delta-9-tetrahydrocannabinol (THC) is increasingly used for the long-term treatment of nausea, vomiting, cachexia, and chronic pain. Recent reports, however, have indicated an increased risk of myocardial infarction and thromboangiitis obliterans after THC intake. Blood platelets have an essential role in the pathogenesis of these two diseases, but it is unclear whether platelets are potential target cells for cannabinoids. We investigated the effects of THC on human platelets and the expression of cannabinoid receptors on their cell membranes in this in vitro study. The effects of THC (final concentrations 10(-7) to 10(-5) M) on the expression of activated platelet fibrinogen receptor (glycoprotein IIb-IIIa) and P selectin were characterized by flow cytometry. Western blotting was performed with platelet membrane preparations to determine the surface expression of cannabinoid receptors on human platelets. THC increased the expression of glycoprotein IIb-IIIa and P selectin on human platelets in a concentration-dependent manner. The two known cannabinoid receptors (CB(1) and CB(2)) were both detected on the cell membrane of human platelets. Our functional results may suggest a receptor-dependent pathway of THC-induced platelet activation. However, further in vivo studies are warranted to evaluate the role of cannabinoid receptors in mediating the demonstrated procoagulatory effect of THC.