Osteoporosis and its Association with Vitamin D Receptor, Oestrogen α Receptor, Parathyroid Receptor and Collagen Type I alpha Receptor Gene Polymorphisms with Bone Mineral Density: A Pilot Study from South Indian Postmenopausal Women of Tamil Nadu.

The involvement of many putative genetic factors makes osteoporosis a complex disease. With increasing longevity of the Indian population, it's now being realized that, as within the West, osteoporotic fractures are also a significant explanation for morbidity and mortality in postmenopausal women. Studies have suggested that the genetic component liable for bone mass could be linked to single nucleotide polymorphisms. Therefore, this study is aimed to research the role of seven gene polymorphisms previously associated with bone phenotype in a cohort of postmenopausal South Indian women from Tamil Nadu. The subjects for the study (n = 300) included 100 osteoporotic women (age 59.3 ± 9.26), 100 osteopenic women (age 55.6 ± 8.17) and 100 non-osteoporotic women as controls (age 55.4 ± 8.85).Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Case-control genetic association analysis of BsmI of the VDR and BstBI of the PTH gene showed a significant allelic association with low bone mineral density amongst the osteoporotic postmenopausal women. The association of BMD with the VDR gene polymorphisms revealed that the average BMD in the BsmI polymorphism with the recessive genotype GG in osteoporotic women was significantly reduced compared with the average BMD in osteoporotic women with AA and AG genotypes. In the BstBI polymorphism, the BMD in the osteoporotic subjects were significantly lower in the AA group than in the GA and GG groups. These results provide evidence for an independent association between BMD and rs1544410 in VDR and rs6254 in PTH and may contribute in being a possible genetic marker for predicting the disease susceptibility in the population tested.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

Colchicine inhibits ROS generation in response to glycoprotein VI stimulation.

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y12 receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H2DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO+/CD62P+) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.

The collagen receptor glycoprotein VI promotes platelet-mediated aggregation of ß-amyloid.

Cerebral amyloid angiopathy (CAA) and ß-amyloid (Aß) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aß aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aß40 to the fibrinogen receptor integrin αIIbß3 Here, we investigated the contribution of the collagen receptor GPVI (glycoprotein VI) in platelet-induced amyloid aggregation. Using platelets isolated from GPVI-wild type and GPVI-deficient human donors and mice, we found that Aß40 bound to GPVI, which induced the release of ATP and fibrinogen, resulting in platelet aggregation. Binding of Aß40 to integrin αIIbß3, fibrinogen, and GPVI collectively contributed to the formation of amyloid clusters at the platelet surface. Consequently, blockade of αIIbß3 or genetic loss of GPVI reduced amyloid fibril formation in cultured platelets and decreased the adhesion of Aß-activated platelets to injured carotid arteries in mice. Application of losartan to inhibit collagen binding to GPVI resulted in decreased Aß40-stimulated platelet activation, factor secretion, and platelet aggregation. Furthermore, the application of GPVI- or integrin-blocking antibodies reduced the formation of platelet-associated amyloid aggregates. Our findings indicate that Aß40 promotes platelet-mediated amyloid aggregation by binding to both GPVI and integrin αIIbß3 Blocking these pathways may therapeutically reduce amyloid plaque formation in cerebral vessels and the brain parenchyma of patients.

Ligand Binding to the Collagen VI Receptor Triggers a Talin-to-RhoA Switch that Regulates Receptor Endocytosis.

Capillary morphogenesis gene 2 (CMG2/ANTXR2) is a cell surface receptor for both collagen VI and anthrax toxin. Biallelic loss-of-function mutations in CMG2 lead to a severe condition, hyaline fibromatosis syndrome (HFS). We have here dissected a network of dynamic interactions between CMG2 and various actin interactors and regulators, describing a different behavior from other extracellular matrix receptors. CMG2 binds talin, and thereby the actin cytoskeleton, only in its ligand-free state. Extracellular ligand binding leads to src-dependent talin release and recruitment of the actin cytoskeleton regulator RhoA and its effectors. These sequential interactions of CMG2 are necessary for the control of oriented cell division during fish development. Finally, we demonstrate that effective switching between talin and RhoA binding is required for the intracellular degradation of collagen VI in human fibroblasts, which explains why HFS mutations in the cytoskeleton-binding domain lead to dysregulation of extracellular matrix homeostasis.

Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS-independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1-mediated BCR phosphorylation on Tyr177, which is important for maintaining ß-catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient-derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.

Generation of Induced Pluripotent Stem Cells from Patients with COL3A1 Mutations and Differentiation to Smooth Muscle Cells for ECM-Surfaceome Analyses.

Use of experimentally derived induced pluripotent stem cells (iPSCs) has led to the development of cell models for differentiation, drug testing and understanding disease pathogenesis. For these models to be informative, reprogrammed cell lines need to be adequately characterized and shown to preserve all of the critical characteristics of pluripotency and differentiation. Here, we report a detailed protocol for the generation of iPSCs from human fibroblasts containing mutations in COL3A1 using a Sendai virus mediated integration-free reprogramming approach. We describe how to characterize the putative iPSCs in vivo and in vitro to ensure potency and differentiation potential. As an example of how these mutations may affect cell surface and extracellular matrix (ECM) interactions, we provide protocols for the differentiation of these cells into smooth muscle cells to illustrate how different cell types may display cell autonomous differences in collagen receptors that may affect their phenotype. These cells, when applied to mechanical model systems (see Chapter 18 by Bose et al.) facilitate an assessment of stiffness and stress-strain relationships useful for understanding how extracellular matrix dysfunction and its interactions with surface proteins contribute to disease processes.

Collagen IV diseases: A focus on the glomerular basement membrane in Alport syndrome.

Alport syndrome is the result of mutations in any of three type IV collagen genes, COL4A3, COL4A4, or COL4A5. Because the three collagen chains form heterotrimers, there is an absence of all three proteins in the basement membranes where they are expressed. In the glomerulus, the mature glomerular basement membrane type IV collagen network, normally comprised of two separate networks, α3(IV)/α4(IV)/α5(IV) and α1(IV)/α2(IV), is comprised entirely of collagen α1(IV)/α2. This review addresses the current state of our knowledge regarding the consequence of this change in basement membrane composition, including both the direct, via collagen receptor binding, and indirect, regarding influences on glomerular biomechanics. The state of our current understanding regarding mechanisms of glomerular disease initiation and progression will be examined, as will the current state of the art regarding emergent therapeutic approaches to slow or arrest glomerular disease in Alport patients.

Thrombosis and von Willebrand Factor.

One of the key players in both hemostasis and thrombosis is von Willebrand factor (vWF), which demonstrates a duality between these two processes. Thrombus is structured by numerous elements, including endothelial cells, platelets, plasma proteins and shear stress alteration. In circulation, once a vessel wall is injured, collagen is exposed and platelets attach to the site of injury. Accordingly, vWF mediates adherence of platelets to the damaged vessel wall by binding both to the collagen and platelet receptor. A growing body of data also indicates a role for neutrophil extracellular traps (NETs) in human thrombosis as scaffolds for vWF, promoting thrombosis. VWF also mediates the protection of factor VIII, a main cofactor of the intrinsic clotting pathway. Since vWF plays a critical role in both thrombotic and bleeding events, a decreased plasma level of this factor may point to a bleeding disorder, while an elevated plasma level may predict occurrence of thrombosis. Since thrombotic events are the foremost cause of death, inhibiting the vWF activity would be a novel prophylaxis to reduce these events. Though, accumulated data have made vWF a promising focus for research on cardiovascular diseases (CVD). This chapter, however, aims to clarify the role of vWF in thrombus formation and pathogenesis of thrombosis.

Stromal Integrin α11ß1 Affects RM11 Prostate and 4T1 Breast Xenograft Tumors Differently.

PURPOSE: It has been implied that the collagen binding integrin α11ß1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11ß1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model. METHODS: Tumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections. RESULTS: RM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups. CONCLUSION: Deficiency of stromal integrin α11ß1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis.

Expression and crystallographic studies of the ligand-binding region of the human endocytic collagen receptor uPARAP.

Urokinase plasminogen activator receptor-associated protein (uPARAP) is an endocytic receptor that internalizes collagen for lysosomal degradation and plays an important role in matrix remodelling. Previous recombinant protein production of uPARAP in Pichia pastoris generated protein with highly heterogeneous glycans that was prone to proteolytic degradation, resulting in highly twinned crystals. In this study, the uPARAP ligand-binding region was expressed in stably transfected Drosophila S2 insect cells. The recombinant protein was homogeneous after purification by metal-affinity and anion-exchange chromatography. Crystals were obtained at two different pH values (5.3 and 7.4) and diffracted to 2.44 and 3.13 Å resolution, respectively. A model of the ligand-binding region of uPARAP was obtained by molecular replacement combined with autobuilding. As the first multidomain crystal structure of the mannose receptor family, structural characterization of the uPARAP ligand-binding region will provide insight into the pH-induced conformational rearrangements of the mannose receptor family.

Reduced granulation tissue and wound strength in the absence of α11ß1 integrin.

Previous wound healing studies have failed to define a role for either α1ß1 or α2ß1 integrin in fibroblast-mediated wound contraction, suggesting the involvement of another collagen receptor in this process. Our previous work demonstrated that the integrin subunit α11 is highly induced during wound healing both at the mRNA and protein level, prompting us to investigate and dissect the role of the integrin α11ß1 during this process. Therefore, we used mice with a global ablation of either α2 or α11 or both integrin subunits and investigated the repair of excisional wounds. Analyses of wounds demonstrated that α11ß1 deficiency results in reduced granulation tissue formation and impaired wound contraction, independently of the presence of α2ß1. Our combined in vivo and in vitro data further demonstrate that dermal fibroblasts lacking α11ß1 are unable to efficiently convert to myofibroblasts, resulting in scar tissue with compromised tensile strength. Moreover, we suggest that the reduced stability of the scar is a consequence of poor collagen remodeling in α11(-/-) wounds associated with defective transforming growth factor-ß-dependent JNK signaling.

Fibroblast α11ß1 integrin regulates tensional homeostasis in fibroblast/A549 carcinoma heterospheroids.

We have previously shown that fibroblast expression of α11ß1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11+/+) or with a deletion (α11-/-) in integrin α11 gene. Our data show that α11-/-/A549 spheroids are larger than α11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11ß1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11ß1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11ß1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.

Integrin α11ß1: a major collagen receptor on fibroblastic cells.

Integrin α11 is the last addition to the vertebrate integrin family. In this chapter we will summarize some basic facts about this integrin and update with information that has been gained in the last decade. Integrin α11ß1 is a major collagen receptor on a subset of fibroblasts. Extensive characterization of the expression pattern in developing mouse embryos has demonstrated expression restricted to subsets of fibroblasts and a transient expression in odontoblasts, but comprehensive characterization of corresponding expression in adult tissues is still lacking. Mice lacking integrin α11 are dwarfed, primarily due to defective incisor eruption defect, which can be traced back to need for α11 on periodontal ligament fibroblasts during incisor eruption. Separate studies have suggested reduced levels of IGF-1 in mice lacking α11. Analysis of lung cancer has identified α11ß1 as a functional important collagen receptor on carcinoma associated fibroblasts (CAFs) and a number of disease models are awaiting analysis to see the importance of this collagen receptor in pathological models.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-ß.

Transforming growth factor ß (TGF-ß) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-ß entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-ß complex is a crucial step in the regulation of TGF-ß function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-ß complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-ß to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion-dependent mechanism. We identify α11ß1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-ß and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells.

Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors.

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a ß-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice.

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2ß1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2ß1 positively delays renal fibrosis: COL4A3(-/-)/ITGA2(-/-) double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3(-/-)/ITGA2(+/-) and COL4A3(-/-)/ITGA2(+/+) mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3(-/-) Alport mice with normal integrin α2ß1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3(-/-)/ITGA2(+/+) Alport mice, but not in COL4A3(+/+)/ITGA2(-/-) mice. In conclusion, the collagen receptors DDR1 and integrin α2ß1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell-matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.

Gene receptor polymorphism as a risk factor for BMD deterioration in adolescent girls with anorexia nervosa.

Anorexia nervosa is a serious eating disorder that is associated with decreased bone mineral density and greater lifetime risk for fractures. This case-controlled study, analyzed single nucleotide polymorphisms of genes encoding vitamin D receptor, estrogen receptor alpha (ESR1), collagen type I and calcitonin receptor (CTR). Relationships between genotype and body mass index, cycling status and lumbar spine bone mineral density (LBMD) were determined in 40 adolescent girls with anorexia nervosa and 10 age-matched controls. The distribution of CTR-AluI genotypes differed between groups, but this polymorphism was not associated with LBMD Z-score. Distribution of ESR1-XbaI genotypes did not differ between groups, but the AA genotype was associated with decreased LBMD Z-score (≤-1) (OR = 24.79, 95% CI, 1.01-606.08). Carriers of the A allele were more likely to have decreased LBMD Z-scores compared with carriers of the G allele (OR = 4.12, 95% CI, 1.23-13.85, p = 0.022). In conclusion, our study shows that anorexic patients with wild-type genotype ESR-XbaI receptor are in greater risk for decreased BMD in relation to those with the mutated gene. Prompt recognition of these patients is crucial because early administration of the proper therapeutic treatment may contribute to the prevention of adverse sequelae on bone metabolism.

Genetic variation of platelet function and pharmacology: an update of current knowledge.

Platelets are critically involved in atherosclerosis and acute thrombosis. The platelet phenotype shows a wide variability documented by the inherited difference of platelet reactivity, platelet volume and count and function of platelet surface receptors. Several candidate genes have been put into focus and investigated for their functional and prognostic role in healthy individuals and patients with cardiovascular (CV) disease treated with antiplatelet agents. In addition to genetic variation, other clinical, disease-related and demographic factors are important so-called non-genetic factors. Due to the small effect sizes of single nucleotide polymorphisms (SNP) in candidate genes and due to the low allele frequencies of functional relevant candidate SNPs, the identification of genetic risk factors with high predictive values generally depends on the sample size of study cohorts. In the post-genome era new array and bioinformatic technologies facilitate high throughput genome-wide association studies (GWAS) for the identification of novel candidate genes in large cardiovascular cohorts. One of the crucial aspects of platelet genomic studies is the precise definition of a specific clinical phenotype (e.g. stent thrombosis) as this will impact importantly the findings of genomic studies like GWAS. Here, we provide an update on genetic variation of platelet receptors and drug metabolising enzymes under specific consideration of data derived by GWAS. The potential impact of this information and the role in personalised therapeutic concepts will be discussed.

[Characterisation of enucleated cells apoptosis: human platelets and erythrocytes].

Apoptosis is a common mechanism of programmed cell death in virtually all nucleated cells. In spite of the fact that platelets and erythrocytes are the only enucleated cells in mammals they contain most of the apoptosis machinery of other cells and undergo similar apoptotic processes as nucleated cells except those connected with nuclear and chromatin transformation. Here we compare the mechanisms of platelet and erythrocytes apoptosis induced by different stimuli namely, stimulation ofthrombin and collagen receptor (T/C), inhibitor of BclX family proteins (ABT-373) for platelets, tert-butylhydroperoxide (tBH) and calcium ionophore (A-23187) for erythrocytes. Induction of platelet apoptosis by both methods (T/C and ABT-373) lead to strong phosphoetydilserine (PS) externalization, loss of the mitochondrial membrane potential (DeltaPsim), proteolytic cleavage of some cytoskeletal and regulatory proteins, and microparticle (MP) formation. However, there are clear differences between mechanisms of platelet apoptosis induced by TC and ABT-373. T/C induced apoptotic reaction is very fast (reach the maximum at 5 min), whereas ABT-373 induced reaction is more prolonged (first apoptotic evidence appears only after 30 min and reach the maximum after 3 hours). MP formation is much more pronounced in T/C than in ABT-373 stimulated platelets, whereas caspase 3 activation is much more stronger in ABT-373 than in T/C stimulated platelets. The main differences between these two apoptotic pathways are connected with aIIbp3 integrin, which activation appears only after T/C stimulation. For tBH experiments on erythrocytes we established optimal conditions (0.25x1012 cells/L, and strong, 1500 RPM stirring) for elucidation of apoptotic processes and found two independent ways of erythrocytes apoptotic processes; calcium independent, connected with met hemoglobin (metHb) formation (tBH stimulation), and calcium dependent pathway (A-23187 stimulation). Erythrocytes apoptosis induced by tBH is characterized by formation ofmetHb, cell shrinkage, fast (95 % during 3 hours) PS externalization, yield of hemoglobin, probably by vesicle (MP) formation. These cells are transformed to stomatocytes, become highly rigid, and could not be lysed even in pure water. All these reactions are calcium independent. Whereas increase of intracellular calcium concentration by A-23187 connected with formation of exinocytes, less pronounced (17 % during 3 h, 35 % during 15 h) PS externalization and rigidity (lysed in 50 mOsm buffer).