HBSP improves kidney ischemia-reperfusion injury and promotes repair in properdin deficient mice via enhancing phagocytosis of tubular epithelial cells.

Phagocytosis plays vital roles in injury and repair, while its regulation by properdin and innate repair receptor, a heterodimer receptor of erythropoietin receptor (EPOR)/ß common receptor (ßcR), in renal ischaemia-reperfusion (IR) remains unclear. Properdin, a pattern recognition molecule, facilitates phagocytosis by opsonizing damaged cells. Our previous study showed that the phagocytic function of tubular epithelial cells isolated from properdin knockout (PKO) mouse kidneys was compromised, with upregulated EPOR in IR kidneys that was further raised by PKO at repair phase. Here, helix B surface peptide (HBSP), derived from EPO only recognizing EPOR/ßcR, ameliorated IR-induced functional and structural damage in both PKO and wild-type (WT) mice. In particular, HBSP treatment led to less cell apoptosis and F4/80+ macrophage infiltration in the interstitium of PKO IR kidneys compared to the WT control. In addition, the expression of EPOR/ßcR was increased by IR in WT kidneys, and furthered increased in IR PKO kidneys, but greatly reduced by HBSP in the IR kidneys of PKO mice. HBSP also increased PCNA expression in IR kidneys of both genotypes. Moreover, iridium-labelled HBSP (HBSP-Ir) was localized mainly in the tubular epithelia after 17-h renal IR in WT mice. HBSP-Ir also anchored to mouse kidney epithelial (TCMK-1) cells treated by H2O2. Both EPOR and EPOR/ßcR were significantly increased by H2O2 treatment, while further increased EPOR was showed in cells transfected with small interfering RNA (siRNA) targeting properdin, but a lower level of EPOR was seen in EPOR siRNA and HBSP-treated cells. The number of early apoptotic cells was increased by EPOR siRNA in H2O2-treated TCMK-1, but markedly reversed by HBSP. The phagocytic function of TCMK-1 cells assessed by uptake fluorescence-labelled E.coli was enhanced by HBSP dose-dependently. Our data demonstrate for the first time that HBSP improves the phagocytic function of tubular epithelial cells and kidney repair post IR injury, via upregulated EPOR/ßcR triggered by both IR and properdin deficiency.

Severe COVID-19 is associated with hyperactivation of the alternative complement pathway.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by impaired type I interferon activity and a state of hyperinflammation leading to acute respiratory distress syndrome. The complement system has recently emerged as a key player in triggering and maintaining the inflammatory state, but the role of this molecular cascade in severe COVID-19 is still poorly characterized. OBJECTIVE: We aimed at assessing the contribution of complement pathways at both the protein and transcriptomic levels. METHODS: To this end, we systematically assessed the RNA levels of 28 complement genes in the circulating whole blood of patients with COVID-19 and healthy controls, including genes of the alternative pathway, for which data remain scarce. RESULTS: We found differential expression of genes involved in the complement system, yet with various expression patterns: whereas patients displaying moderate disease had elevated expression of classical pathway genes, severe disease was associated with increased lectin and alternative pathway activation, which correlated with inflammation and coagulopathy markers. Additionally, properdin, a pivotal positive regulator of the alternative pathway, showed high RNA expression but was found at low protein concentrations in patients with a severe and critical disease, suggesting its deposition at the sites of complement activation. Notably, low properdin levels were significantly associated with the use of mechanical ventilation (area under the curve = 0.82; P = .002). CONCLUSION: This study sheds light on the role of the alternative pathway in severe COVID-19 and provides additional rationale for the testing of drugs inhibiting the alternative pathway of the complement system.

Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia-Reperfusion.

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia-reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Properdin Is a Modulator of Tumour Immunity in a Syngeneic Mouse Melanoma Model.

Background and Objectives: Tumours are often low immunogenic. The role of complement, an innate immune defence system, in tumour control has begun to be elucidated, but findings are conflicting. A role for properdin, an amplifier of complement activation, in tumour control has recently been implicated. Materials and Methods: Properdin-deficient and congenic wildtype mice were injected subcutaneously with B16F10 melanoma cells. Tumour mass and chemokine profile were assessed. The frequencies of CD45+CD11b+ Gr-1+ cells were determined from tumours and spleens, and CD206+ F4/80+ cells were evaluated in spleens. Sera were analysed for C5a, sC5b-9, and CCL2. Results: Whilst there was no difference in tumour growth at study endpoint, properdin-deficient mice had significantly fewer myeloid-derived suppressor cells (MDSCs) in their tumours and spleens. Splenic M2 type macrophages and serum levels of C5a, sC5b-9, and CCL2 were decreased in properdin-deficient compared to wildtype mice. Conclusions: The presence of intact complement amplification sustains an environment that lessens potential anti-tumour responses.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Compound heterozygous mutations in IL10RA combined with a complement factor properdin mutation in infantile-onset inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel diseases (IBDs) are chronic and multifactorial diseases resulting from a complex interaction of host genetic factors and environmental stimuli. Although many genome-wide association studies have identified host genetic factors associated with IBD, rare Mendelian forms of IBD have been reported in patients with very early onset forms. Therefore, this study aimed to identify genetic variants associated with infantile-onset IBD. PARTICIPANTS AND METHODS: We obtained genomic DNA from whole blood samples of a male patient with infantile-onset IBD and nonconsanguineous Korean parents. Whole-exome sequencing was performed using trio samples. Then, we analyzed the data using susceptibility genes for monogenic forms of IBD and various immunodeficiencies and protein structural analysis. RESULTS: The patient who presented with oral aphthous ulcers at the age of 14 days suffered from severe colitis and was refractory to medical treatment. Compound heterozygous mutations in IL10RA (p.R101W; p.T179T) were found in the patient. In addition, a hemizygous mutation in complement factor properdin (CFP) (p.L456V) located on the X-chromosome was detected, inherited from the patient's mother. Protein structural modeling suggested impaired properdin subunit interactions by p.L456V that may hamper protein oligomerization required for complement activation. CONCLUSION: This study identified compound heterozygous mutations in IL10RA combined with a hemizygous CFP mutation in infantile-onset IBD by using whole-exome sequencing. CFP p.L456V may exacerbate symptoms of infantile-onset IBD by disturbing oligomerization of properdin.

Human Properdin Opsonizes Nanoparticles and Triggers a Potent Pro-inflammatory Response by Macrophages without Involving Complement Activation.

Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.

Experimentally-induced anti-myeloperoxidase vasculitis does not require properdin, MASP-2 or bone marrow-derived C5.

Anti-neutrophil cytoplasmic antibody vasculitis is a systemic autoimmune disease with glomerulonephritis and pulmonary haemorrhage as major clinical manifestations. The name reflects the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind to both neutrophils and monocytes. Evidence of the pathogenicity of these autoantibodies is provided by the observation that injection of anti-myeloperoxidase antibodies into mice causes a pauci-immune focal segmental necrotizing glomerulonephritis which is histologically similar to the changes seen on renal biopsy in patients. Previous studies in this model have implicated the alternative pathway of complement activation and the anaphylatoxin C5a. Despite this progress, the factors that initiate complement activation have not been defined. In addition, the relative importance of bone marrow-derived and circulating C5 is not known. This is of interest given the recently identified roles for complement within leukocytes. We induced anti-myeloperoxidase vasculitis in mice and confirmed a role for complement activation by demonstrating protection in C3-deficient mice. We showed that neither MASP-2- nor properdin-deficient mice were protected, suggesting that alternative pathway activation does not require properdin or the lectin pathway. We induced disease in bone marrow chimaeric mice and found that circulating and not bone marrow-derived C5 was required for disease. We have therefore excluded properdin and the lectin pathway as initiators of complement activation and this means that future work should be directed at other potential factors within diseased tissue. In addition, in view of our finding that circulating and not bone marrow-derived C5 mediates disease, therapies that decrease hepatic C5 secretion may be considered as an alternative to those that target C5 and C5a. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Properdin provides protection from Citrobacter rodentium-induced intestinal inflammation in a C5a/IL-6-dependent manner.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium-induced colitis. Following infection, properdin knockout (P(KO)) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell-derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to P(KO) mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of P(KO) mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

Complement gene expression in human cardiac allograft biopsies as a correlate of histologic grade of injury.

Complement activation contributes to antibody-mediated allograft rejection, but increasing evidence also implicates complement proteins produced locally within the graft, in part by infiltrating mononuclear cells, as important mediators of tissue injury. To test this concept in transplant recipients, we evaluated complement, complement regulator, and T cell/proinflammatory marker gene expression by quantitative real-time polymerase chain reaction in 71 archived heart transplant biopsies and correlated the results with the histologic grade of rejection. Significantly more transcripts encoding alternative pathway components factor B, C3 and properdin, and C3a receptor and C5a receptor were detected in grade 3 versus grade 0 or 1 biopsies. The grade 3 rejections also contained significantly higher amounts of CD3, interferon gamma, perforin, and granzyme B genes. In addition to providing supportive evidence for a pathogenic role of graft-derived complement in human heart transplant injury, these correlations suggest that molecular profiling of complement gene expression could be useful in the diagnosis of human allograft rejection.

Three isoforms of complement properdin factor P in trout: cloning, expression, gene organization and constrained modeling.

Properdin is a plasma glycoprotein and the only known naturally occurring positive regulator of the complement system, stabilizing the alternative pathway convertase (C3bBb). In order to elucidate the molecular evolution of properdin factor P (pfc), here we report the cloning and characterization of three gene isoforms of properdin in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout properdins pfc1, pfc2 and pfc3 (447, 449 and 447 amino acids, respectively) share 78-90% identity to each other, showing the highest identity score (47%) with their zebrafish ortholog protein. The overall identity with human, mouse and xenopus properdin polypeptides is 44%, 42% and 45%, respectively. The 'domain' architecture of trout properdins resembles that of the mammalian counterpart proteins, composed of six thrombospondin repeat type 1 domains (TSR-1-TSR-6). TSR domains of the three trout properdin isoforms seem to adopt the folding pattern of human thrombospondin 1 TSP-1 domains, where each TSP-1 domain forms an antiparallel three-stranded structure that consists of alternative stacked layers of Trp and Arg residues from respective strands capped by disulfide bonds on each end. The trout pfc2 and pfc3 genes are arranged in nine and ten exons, respectively, which span approximately 3.5kb of the genome. In contrast to the expression profile of the properdin gene in mammals, liver is the main source of the trout properdin mRNA transcripts. In a phylogenetic analysis, trout pfc1, pfc2 and pfc3 genes are clustered with their orthologs from other teleost species. This is the first report of three separate genes coding for properdin factor P in a vertebrate species.

Serum amyloid A, properdin, complement 3, and toll-like receptors are expressed locally in human sinonasal tissue.

BACKGROUND: There is a growing appreciation of the role that nasal mucosa plays in innate immunity. In this study, the expression of pattern recognition receptors known as toll-like receptors (TLRs) and the effector molecules complement factor 3 (C3), properdin, and serum amyloid A (SAA) were examined in human sinonasal mucosa obtained from control subjects and patients with chronic rhinosinusitis (CRS). METHODS: Sinonasal mucosal specimens were obtained from 20 patients with CRS and 5 control subjects. Messenger RNA (mRNA) was isolated and tested using Taqman real-time polymerase chain reaction with primer and probe sets for C3, complement factor P, and SAA. Standard polymerase chain reaction was performed for the 10 known TLRs. Immunohistochemistry was performed on the microscopic sections using antibodies against C3. RESULTS: Analysis of the sinonasal sample mRNA revealed expression of all 10 TLRs in both CRS samples and in control specimens. Expression of the three effector proteins was detected also, with the levels of mRNA for C3 generally greater than SAA and properdin in CRS patients. No significant differences were found in TLR or innate immune protein expression in normal controls. Immunohistochemical analysis of sinonasal mucosal specimens established C3 staining ranging from 20 to 85% of the epithelium present. CONCLUSION: These studies indicate that sinonasal mucosa expresses genes involved in innate immunity including the TLRs and proteins involved in complement activation. We hypothesize that local production of complement and acute phase proteins by airway epithelium on stimulation of innate immune receptors may play an important role in host defense in the airway and, potentially, in the pathogenesis of CRS.

Increased levels of acylation-stimulating protein in interleukin-6-deficient (IL-6(-/-)) mice.

IL-6-deficient (IL-6(-/-)) mice develop obesity at 6-7 months of age. To elucidate the mechanisms of this mature-onset obesity, global gene expression profiles of 3-month-old preobese IL-6(-/-) were compared with those of IL-6(+/+) mice using DNA arrays. Genes that were up-regulated in IL-6(-/-) mice included the factors transthyretin and properdin in white adipose tissue and adipsin in muscle. These factors have been shown to influence the formation of acylation-stimulating protein (ASP), a cleavage product of complement C3. ASP stimulates the synthesis of triacylglycerol in adipocytes, and ASP-deficient mice are resistant to diet-induced obesity. In line with the increases in transthyretin, properdin, and adipsin, ASP levels in serum were increased by 31-54% in IL-6(-/-) compared with IL-6(+/+) mice. Furthermore, IL-6 replacement treatment in IL-6(-/-) mice decreased ASP levels significantly by 25-60%. In conclusion, ASP levels are increased in preobese IL-6(-/-) mice. This increase may result in increased triacylglycerol formation and uptake in IL-6(-/-) adipocytes and thereby contribute to the development of obesity in IL-6(-/-) mice.

Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin.

Properdin (P) is a serum glycoprotein that stabilizes the labile C3 convertase (C3bBb) of the alternative pathway of the complement system (AP). Thanks to its oligomeric nature, P specifically upregulates AP on surfaces without activating AP in the fluid-phase. We investigated whether human cells, displaying P at their membrane, could activate autologous AP. The cDNAs encoding human P and the transmembrane domain of human platelet derived growth factor receptor were fused together and expressed in human embryo kidney cells (HEK-293). Selected cells displayed P at their surface as shown by FACS. In contact with human serum at 37 degrees C, they triggered AP-mediated C3 deposition. SDS-PAGE analysis showed C3 covalently bound to various membrane proteins, but not to P itself. However, displayed P affinity could bind to serum or purified C3i at 4 degrees C. C3 binding was restricted to the cells displaying P, was inhibited by an anti-P mAb, and did not require serum P. Bound C3 allowed further C5, C7 and C9 deposition as well as cell lysis after blocking CD59 function. In contrast, wild-type cells, cells displaying factor D or truncated P (deleted from its 6th thrombospondin-like repeat) did not activate AP. We hypothesize that displayed P activates AP by stabilizing bystander C3b and/or by capturing serum C3iBb convertase. Finally, we suggest that P could be used for retargeting autologous complement to AP-resistant pathogens and tumor cells.

Cloning and characterization of the cDNA encoding guinea-pig properdin: a comparison of properdin from three species.

The cDNA sequence encoding properdin was generated from guinea-pig spleen RNA by the reverse transcription-polymerase chain reaction. This sequence was approximately 75% homologous with human and 71% homologous with murine properdin at the nucleic acid level. Guinea-pig properdin had six thrombospondin repeat sequences consisting of about 60 amino acids, each with six cysteine and three tryptophan residues. Additionally, the Valine-Threonine-Cysteine-Glycine sequence, reported to have important cell adhesive properties in malarial circumsporozoite proteins and thrombospondin, was conserved in the properdin sequence of guinea-pigs. Finally, mouse spleen was also examined to complete the sequence determination of the leader peptide and the initial four residues of murine properdin. This allowed a thorough comparison of the primary structure of properdin from all three species. Like human and murine properdin cDNAs, the guinea pig sequence contained a region of unique, non-homologous sequence (18 base pairs in length) within the fifth thrombospondin repeat, the significance of which remains unclear.

Expression of properdin in human monocytes.

Properdin is the only known positive regulator of the alternative pathway of complement activation. Northern blot analysis of cell lines derived from fibroblasts, B-cells, hepatoma cells, and cells of the monocyte-macrophage lineage revealed properdin expression only in the myelomonocytic cell line HL-60, in the monoblastic cell line U-937 and in the monocytic line Mono Mac 6. Culture of Mono Mac 6 cells for 24 h with phorbol 12-myristate 13-acetate, bacterial lipopolysaccharide and the cytokines interleukin-1 beta and tumour necrosis factor-alpha enhanced mRNA abundance, with the strongest effect (tenfold) being observed with the lipopolysaccharide. In contrast, recombinant interferon-gamma consistently halved properdin mRNA abundance. The same pattern was found for the secretion of properdin as detected by ELISA of Mono Mac 6 supernatants. The suppressive effect of interferon-gamma on properdin mRNA abundance was also demonstrated for primary blood monocytes. The data suggest that the expression and secretion of this complement regulatory protein by monocytes is differentially regulated by cytokines and link the immune response with alternative pathway activation.

Convergent evolution: the need to be explicit.

Convergence as a phenomenon in molecular evolution is an issue that confuses many discussions. Often the problem is that not enough care is taken to state exactly what kind of convergence one has in mind. Functional and mechanistic convergence are both common, and some structural convergence has probably occurred, but a convincing case for genuine sequence convergence has yet to be made.

Physical linkage of the A-raf-1, properdin, synapsin I, and TIMP genes on the human and mouse X chromosomes.

Genes encoding the neuron-specific phosphoprotein synapsin I (SYN1), the glycoprotein tissue inhibitor of metalloproteinases (TIMP), the proto-oncogene A-raf-1 (ARAF1), and properdin (PFC), a positive regulator of the alternative pathway of human complement, lie within a conserved synteny encompassing the proximal short arm of the human X chromosome (Xp21.1-p11) and the centromeric end of the mouse X chromosome (A1-A5). We have used a mouse interspecific cross to demonstrate genetic linkage of Syn-1, Timp, and Araf and also show physical linkage, with Timp lying only 10 kb from Araf, within an intron of the Syn-1 gene. Detailed restriction mapping shows that Timp is transcribed in the same direction as Araf but in the opposite direction to the Syn-1 gene. Analysis of the corresponding region of the human X chromosome indicates a similar arrangement and in addition shows that the properdin gene lies within 5 kb of the 5' end of the synapsin I gene.

Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins.

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.