Turnover in humans of β2-microglobulin: the constant chain of HLA-antigens

Abstract The turnover of β₂-microglobulin, the common subunit of the HLA antigens, has been examined in normal subjects and in some patients with kidney disorders, multiple myeloma and rheumatoid arthritis. All patients displayed elevated serum levels of β₂-microglobulin. The plasma disappearance curve of ¹²⁵I-β₂-microglobulin demonstrated that the protein has a rapid turnover (11/2 = 21 h; range 1 1–2–8 h) in normal persons and in patients with a normal glomerular filtration rate. In patients with kidney disorders the impaired renal filtration prolonged the turnover time and led to elevated serum levels of β₂-microglobulin.
Simultaneous measurements of ¹²⁵-β₂-microglobulin in serum and urine allowed estimations of the β₂-microglobulin net reabsorption in the renal tubuli. Two patients with renal disease reabsorbed 84% and 89%, respectively, of the β₂-microglobulin filtered in the glomeruli. In normal persons the net reabsorption is close to 100%.
In patients with normal kidney function increased serum levels of β₂-microglobulin seem to be due to an increased synthetic rate of the protein as the elimination rate is normal. HLA antigen heavy chains in serum are present in smaller amounts than β₂-microglobulin. The present data, therefore, suggest an imba-lanced synthesis of the two chains.     

Serum β2-microglobulin at remission and relapse in patients with multiple myeloma

Abstract. Serum β₂-microglobulin (S-β₂m) was determined before treatment in fifty patients with multiple myeloma (MM), twenty-two of which had pure Bence Jones (BJ) myeloma. S-β₂m was related to clinical stage before, but not after, a correction of S-β₂m values for co-existent raised S-creatinine values (> 106 μmol 1^-1)- However, S-β₂m as well as corrected β₂m was a parameter of prognostic value. The median expected survival time was 14 months at S-β₂m values > 8·0 mg 1^-1 and 56 months at values<3·5 mg 1^-1. A response to treatment was associated with a decrease of S-β₂m and a stationary course with unchanged values, whereas a relapse or progressive disease was connected with an increase. In β₂m producers', i.e. patients with a clear initial high S-β₂m, serial determinations are of value for monitoring patients with MM. In particular, this is the case in BJ myelomas, as they lack a quantifiable serum M-component. With respect to β₂m no difference was found between BJ and other patients with MM.     

Kidney function and size in normal subjects before and during growth hormone administration for one week

Abstract. Kidney function and size were studied in seven normal male subjects before and after administration of highly purified human growth hormone for 1 week. Glomerular filtration rate, renal plasma flow (steady-state infusion technique with urinary collections using ¹²⁵I-iothalamate and ¹³¹I-hippuran) kidney size (ultrasonic scanning) and urinary excretion rates of albumin and β₂-microglobulin (radioimmunoas-says) were measured. Highly purified growth hormone was injected subcutaneously, 2 IU in the morning and 4 IU in the evening. Glomerular filtration rate increased from (mean ± SEM) 114 ± 5 to 125±4ml/min x 1.73 m² ( P <0.01) and renal plasma flow increased from 554 ±30 to 601 ±36 ml/min ×1.73 m²( P < 0.01). Kidney size and urinary excretion rates of albumin and β₂-microglobulin did not change significantly.
Our results show that raising plasma growth hormone into a range similar to that found in insulin-dependent diabetics enhances glomerular filtration rate and renal plasma flow, while kidney size remains unchanged. Increased renal plasma flow is the major determinant of growth hormone induced elevation in glomerular filtration rate. Growth hormone may thus contribute to the enhancement of glomerular filtration rate and renal plasma flow typically found in insulin-dependent diabetics.     

Pharmacological characteristics and species-related variations of β3-adrenergic receptors

Summary— Beta-adrenergic receptors (β/-AR) belong to the large multigenic family of receptors coupled to GTP-binding proteins. Three subtypes have been identified: β₁-, β₂- and β₃-AR. Much of the work delineating the precise pharmacological comparison of the three β-ARs has come from investigations with stably transfected Chinese hamster ovary cells (CHO cells). This review discusses the structure and function of β₃-AR in various species and presents new findings on a number of β₃-AR ligands including carazolol, tertatolol and CL 316,243 which were found to be selective and potent β₃-AR agonists and ZD 2079 and salmeterol which appear to display full but non-subtype selective agonistic activity. Species-related variations of the β₃-AR pharmacology have been shown for propranolol and bupranolol. With the ongoing characterization of the β₃-AR at the molecular and cellular level, and with the advent of computer-assisted molecular modelling to aid in the determination of the three-dimensional structure of the receptor, it is thought that novel β₃-AR compounds will become available with improved selectivity and potency.     

A simple method to discriminate between β2-glycoprotein I- and prothrombin-dependent lupus anticoagulants

Summary.  Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against β₂-glycoprotein I (β₂GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against β₂GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between β₂GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-β₂GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-β₂GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on β₂GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of β₂GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-β₂GPI antibodies of the CL-induced prolongation of the PTT-LA.     

Relationship between fractional calcium absorption and gastric emptying

Abstract. The relationship between calcium absorption and gastric emptying and the precision of measurement of fractional calcium absorption using a single isotope technique were evaluated in 14 normal postmenopausal women (age range 61–72 years). On two occasions separated by between 5 and 15 days, each subject was given 250 mL water containing 0.2 MBq of ⁴⁵Ca in 20 mg of calcium carrier as the chloride, 20 mg kg^-1 paracetamol and 9 MBq of ^99mTc sulphur colloid. Venous blood samples were taken at -2, 15, 30, 45, 60, 90, 120, 150 and 180 min after consumption of the drink, and gastric emptying (GE) was monitored with a gamma camera. Fractional calcium absorption in the first hour (α₆) was calculated from the blood samples obtained at 15, 30, 45, 60, 90 and 120 min. An absorption rate was also derived from the 60 min sample using only a calibration curve (α₁). There were close correlations between radiocalcium absorption on the two study days ( r = 0.89, P < 0.001 for both α₁ and α₆) and between α₁ and α₆ ( r = 0.93, P < 0.001). Plasma paracetamol concentrations at 15 min were directly related to the early phase of GE ( r = 0.42, P < 0.05). In contrast, calcium absorption was inversely related to GE ( r — 0.45, P < 0.05). We conclude that radiocalcium absorption is not greatly influenced by gastric emptying rate and that the single blood sample procedure has similar precision to the six-blood sample test.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Urinary excretion of bile alcohols in normal children and patients with α1-antitrypsin deficiency during development of liver disease

Abstract. Healthy infants and children were found to excrete bile alcohol glucuronides in urine. Following isolation and hydrolysis, the bile alcohols were estimated by capillary gas-liquid chromatography. The daily urinary excretion of the major compound, 27-nor-5 β -cholestane-3α,7α,12α,24ξ,25ξ-pentol (a C₂₆ bile alcohol), ranged from 0·1 to 1·1 μmol/24 h per m² body surface area for healthy infants and children. Two groups of patients with α₁-antitrypsin deficiency (phenotype PiZ) were also studied during infancy and childhood, and biochemical liver function tests and liver morphology were compared to the excretion of bile alcohols. The highest excretion of the C₂₆ bile alcohol in urine was found in patients with α₁-antitrypsin deficiency and juvenile cirrhosis (2·1–8·4 μmol 24 h^-1 m^-2) regardless of preceding neonatal cholestasis. Patients with α₁-antitrypsin deficiency, neonatal cholestasis and subsequent fibrosis or normal liver morphology excreted bile alcohols within the normal range. The C₂₆ bile alochol constituted an average of 36% of the total bile alcohols in forty-three urine samples. This percentage was about the same in the three groups studied. The findings suggest that determination of urinary bile alcohols may be a valuable non-invasive diagnostic tool for patients with or at risk of developing liver cirrhosis.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.     

Alzheimer amyloid β-peptides exhibit ionophore-like properties in human erythrocytes

Abstract. There is growing evidence that the amyloid β-peptide (β₁_₄₀) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta₁_₄₀ has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether β₁-₄₀ (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2h and 4h at 37°C together with 6μmolL^-1 of β₁-₄₀ or of fragments β₁_₂₈and β₂₅-₃₅, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05μmolL^-1). Moreover, β₁_₄₀ and its two fragments induced a significant alteration of ⁴⁵Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of β₁_₄₀ and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca²⁺-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease.     

Autoimmune antiphospholipid antibodies impair the inhibition of activated factor X by protein Z/protein Z-dependent protease inhibitor

Summary.  The hemostatic process is tightly regulated by several antithrombotic mechanisms. Among them, protein Z (PZ)-dependent protease inhibitor (ZPI) potently inhibits factor (F)Xa in a manner dependent on calcium ions, phospholipids and PZ. Autoimmune antiphospholipid antibodies (aPL) are mainly directed against phospholipid-binding plasma proteins such as β₂-glycoprotein I (β₂GPI) and prothrombin, and are known to interfere with phospholipid-dependent hemostatic pathways. In this study, we investigated whether purified aPL are able to interfere with inhibition of FXa by PZ/ZPI. β₂GPI modestly delayed the FXa inactivation by PZ/ZPI and most isolated aPL-IgGs were found to further increase the inhibitory potential of β₂GPI on PZ/ZPI activity. Without β₂GPI, the PZ/ZPI activity was unaffected by the addition of aPL-IgG. As PZ deficiency is hypothesized to lead to a prothrombotic state, we performed a case-control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in the 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%) compared with the normal group (4.6%, P < 0.001 vs. APS). ZPI antigen levels were similar in patients with aPL and normal controls. Concomitant PZ deficiency increased by approximately sevenfold the risk of arterial thrombosis in aPL patients. Taken together, these data suggest that the PZ/ZPI system is commonly impaired in aPL patients thus probably increasing the thrombotic risk.     

Hypertriglyceridaemia Secondary to Liver Disease

Abstract. Some patients with liver dysfunction show a marked hypertriglyceridaemia, a phenomenon most frequently accompanied by cholestasis.
In this report we have identified, isolated and characterized an abnormally large (300–700 Å), triglyceride rich, low density lipoprotein, (d 1.019-1.063 g/ml) designated β₂-lipoprotein (β₂-LP), from the plasma of patients with hypertriglyceridaemia secondary to liver disease. The β₂-LP differs significantly in its percent composition and protein moiety from the unique lipoprotein-X (LP-X) specific for cholestasis and also from normal β-lipoproteins, both of which are also present in the patients LDL fraction. Furthermore, some evidence is provided suggesting that the β₂-LP is likely to represent an intermediate particle of chylomicron metabolism, which accumulates in this disease due to a markedly diminished hepatic lipase activity.     

Beta3-adrenoceptors in the cardiovascular system

Summary— Behind the classic beta₁, and beta₂-adrenoceptors, recent molecular and pharmacological studies have described a new receptor, called the beta₃-adrenoceptor, in various mammalian tissues (brown and white adipose tissue, digestive smooth muscle). Few authors have investigated the putative existence of the beta₃-adrenoceptor in the cardiovascular system. This paper reviews the available data. In vitro studies show that beta₃-adrenoceptor agonists (BRL 37344, CGP 12177) induce a relaxation of fragments of rat carotid artery which is not antagonized by propranolol. In dogs, these drugs elicit a decrease in blood pressure due to peripheral vasodilation and an increase in heart rate which is of baroreflex origin. The vasodilating effects are mainly observed in cutaneous and adipose tissue vessels and cannot be explained by any known transductional mechanism. Activation of this vascular β₃-adrenoceptor requires higher doses of catecholamines than for β₁- or β₂-adrenoceptors. In humans, the cardiovascular effects of beta₃-adrenoceptor agonists are explained by the activation of beta₁- or beta₂ (and not beta₃-)-adrenoceptors. These studies suggest the presence of vascular (but not cardiac) beta₃-adrenoceptors in dogs. In other species, including man, the presence of such cardiac β₃-adrenoceptors remains to be resolved. Their physiological relevance remains unknown.     

β2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain

Summary.  Background:  Beta-2 glycoprotein I (β₂GPI) is a plasma glycoprotein which interacts with various proteins of the coagulation and fibrinolysis system. β₂GPI has recently been shown to have anti-angiogenic properties. Objectives:  We undertook this study to investigate the specific domain of β₂GPI involved in the anti-angiogenic function and its effect on downstream signaling of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods:  Various preparations of β₂GPI were used on human umbilical vein endothelial cells (HUVECs) in the absence or presence of VEGF and bFGF. The effect on HUVECs' proliferation, migration and tubule formation in Matrigel matrix was investigated. The effect of β₂GPI on the mRNA expression of VEGF receptors and phosphorylation of signaling molecules was also studied. Results:  β₂GPI is shown in this study to be an anti-angiogenic molecule in vitro by inhibiting VEGF and bFGF-induced proliferation, migration and papillary-like tubule formation of HUVECs. This inhibition was achieved by native, proteolytically clipped and domain deletion mutants, domain I-IV (DI-IV) but not domain II-V (DII-V) of β₂GPI. Native β₂GPI was found to downregulate the expression of the VEGF receptor KDR/Flk-1 on endothelial cells and to block the phosphorylation of VEGF's downstream effector molecules in the MAPK/ERK and PI3K/Akt/GSK3β pathways. Conclusions:  These results indicate that β₂GPI has anti-angiogenic functions which depend on the presence of domain I. This anti-angiogenic activity may have important implications for the therapeutic manipulation of angiogenesis in various disease states.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

CIB1 deficiency results in impaired thrombosis: the potential role of CIB1 in outside-in signaling through integrin αIIbβ3

Summary.  Background: Agonist-induced inside-out signaling activates platelet integrin α_IIbβ₃, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through α_IIbβ₃ regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating α_IIbβ₃ function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl₃-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β₃ subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.