Jaap-Jan Zwaginga

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

P-Selectin Glycoprotein Ligand-1 Is Expressed on Endothelial Cells and Mediates Monocyte Adhesion to Activated Endothelium

Objective—The purpose of this study was to investigate the presence and functionality of P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs). Methods and Results—We show here that PSGL-1 is expressed at the mRNA and protein levels in umbilical vein and microvascular ECs. Furthermore, this endothelial PSGL-1 (ePSGL-1) is functional and mediates adhesion of monocytes or platelet-monocyte complexes (PMCs) to the activated endothelium in a flow model. ePSGL-1 expression was not affected by treating ECs with inflammatory stimuli (tumor necrosis factor α, interleukin-1β, thrombin, or histamine). However, the functional binding capacity of ePSGL-1 to monocytes or P-selectin/Fc chimera significantly increased by stimulation of the ECs with TNFα. By means of a siRNA approach to specifically knock-down the genes involved in the glycosylation of PSGL-1 we could show that tumor necrosis factor α–induced glycosylation of ePSGL-1 is critical for its binding capacity. Conclusion—Our results show that ECs express functional PSGL-1 which mediates tethering and firm adhesion of monocytes and platelets to inflamed endothelium.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Transplantation of human peripheral blood CD34-positive cells in combination with ex vivo generated megakaryocytes results in fast platelet formation in NOD/SCID mice.

Transplantation of human peripheral blood CD34-positive cells in combination with ex vivo generated megakaryocytes results in fast platelet formation in NOD/SCID mice

The (patho)physiology of megakaryocytopoiesis: from thrombopoietin in diagnostics and therapy to ex vivo generated cellular products

The height of the normal level of blood platelets (150–350 × 10 9 /l) and their short life span (approximately 10 days) explain their production rate of 150 × 10 6 /min. Thrombopoietin (Tpo) is the key growth factor in this production (reviewed in [1] ). Tpo consists of a 153 amino acids long amino-terminal receptorbinding domain and a 179 amino acids carboxy-terminal domain, which is involved in its secretion and protection from proteolysis. The Tpo receptor, Mpl, was discovered in 1992 [2] and is expressed on haematopoietic stem cells (HSCs; CD34 + ), the large polyploid megakaryocyte (MK; platelet progenitor) and platelets themselves [3]. Though genetic elimination of Mpl and Tpo in mice was shown to lead to a strong reduction (85%) of platelet numbers (reviewed in [4]), some platelets are still formed. While synergy with Tpo has been described for SCF [5], effective platelet formation might also depend on factors like SDF-1 and FGF-4. Upregulation of adhesion molecules by these factors enhances late megakaryocytic development and platelet formation by growth promoting interactions between MKs and marrow cells [6].