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Importance: Identifying genes and proteins for cognitive resilience (ie, targets that may be associated with slowing or preventing cognitive decline regardless of the presence, number, or combination of common neuropathologic conditions) provides a complementary approach to developing novel therapeutics for the treatment and prevention of Alzheimer disease and related dementias., Objective: To identify proteins associated with cognitive resilience via a proteome-wide association study of the human dorsolateral prefrontal cortex., Design, Setting, and Participants: This study used data from 391 community-dwelling older persons who participated in the Religious Orders Study and the Rush Memory and Aging Project. The Religious Orders Study began enrollment January 1, 1994, and the Rush Memory and Aging Project began enrollment September 1, 1997, and data were collected and analyzed through October 23, 2019., Exposures: Participants had undergone annual detailed clinical examinations, postmortem evaluations, and tandem mass tag proteomics analyses., Main Outcomes and Measures: The outcome of cognitive resilience was defined as a longitudinal change in cognition over time after controlling for common age-related neuropathologic indices, including Alzheimer disease, Lewy bodies, transactive response DNA-binding protein 43, hippocampal sclerosis, infarcts, and vessel diseases. More than 8000 high abundance proteins were quantified from frozen dorsolateral prefrontal cortex tissue using tandem mass tag and liquid chromatography-mass spectrometry., Results: There were 391 participants (273 women); their mean (SD) age was 79.7 (6.7) years at baseline and 89.2 (6.5) years at death. Eight cortical proteins were identified in association with cognitive resilience: a higher level of NRN1 (estimate, 0.140; SE, 0.024; P = 7.35 × 10-9), ACTN4 (estimate, 0.321; SE, 0.065; P = 9.94 × 10-7), EPHX4 (estimate, 0.198; SE, 0.042; P = 2.13 × 10-6), RPH3A (estimate, 0.148; SE, 0.031; P = 2.58 × 10-6), SGTB (estimate, 0.211; SE, 0.045; P = 3.28 × 10-6), CPLX1 (estimate, 0.136; SE, 0.029; P = 4.06 × 10-6), and SH3GL1 (estimate, 0.179; SE, 0.039; P = 4.21 × 10-6) and a lower level of UBA1 (estimate, -0.366; SE, 0.076; P = 1.43 × 10-6) were associated with greater resilience., Conclusions and Relevance: These protein signals may represent novel targets for the maintenance of cognition in old age.

Purpose: The present study was aimed at determining the serum levels of actinin-4 (ACTN4) in cervical cancer (CC) and investigating the diagnostic and prognostic value of serum ACTN4 in CC., Materials and Methods: We included 93 CC patients, 52 cervical intraepithelial neoplasia (CIN) patients, and 70 healthy women. Serum ACTN4 levels were assessed using an ELISA method. A receiver operating characteristic (ROC) curve was performed to evaluate the diagnostic value of serum ACTN4. The survival curves were used to display the overall survival distributions., Results: Serum ACTN4 levels in CC patients were 48.39 ± 13.98 pg/mL which is significantly higher than those in CIN patients (32.72 ± 9.44 pg/mL; P < 0.001) and those in healthy controls (30.84 ± 8.08 pg/mL; P < 0.001). The ROC analysis demonstrated that the area under the curve (AUC) of ACTN4 was 0.852 (95%CI = 0.796-0.908), with sensitivity of 76.3% and specificity of 87.7%. Serum ACTN4 levels were associated with the FIGO stage, lymph node metastasis, and lymphovascular space invasion of CC (all P < 0.05). The survival curve suggested that high serum ACTN4 levels were related to poor prognosis., Conclusion: Our findings suggest that serum ACTN4 levels may be valuable diagnostic and prognostic biomarkers for CC., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright © 2020 Xigui Ma et al.)

Del Coso, J, Salinero, JJ, Lara, B, Gallo-Salazar, C, Areces, F, Herrero, D, and Puente, C. Polygenic profile and exercise-induced muscle damage by a competitive half-ironman. J Strength Cond Res 34(5): 1400-1408, 2020-To date, it is still unknown why some individuals develop higher levels of muscle damage than other individuals, despite participating in exercise with comparable levels of physical intensity. The aim of this investigation was to analyze 7 single-nucleotide polymorphisms (SNPs) that are candidates to explain individual variations in the level of muscle damage attained during a half-ironman competition. Using the model of Williams and Folland (2, 1, and 0 points for optimal, intermediate, and suboptimal genotype), we determined the total genotype score from the accumulated combination of 7 SNPs (ACE = 287bp Ins/Del; ACTN3 = p.R577X; creatine kinase, muscle type = NcoI; insulin-like growth factor 2 = C13790G; interleukin-6 = 174G>C; myosin light chain kinase = C37885A; and tumor necrosis factor-α = 308G>A) in 22 experienced triathletes. Before and after the race, a sample of venous blood was obtained to measure serum markers of muscle damage. Two groups of triathletes were established according to their postcompetition serum CK concentration: low CK responders (n = 10; 377 ± 86 U·L) vs. high CK responders (n = 12; 709 ± 136 U·L). At the end of the race, low CK responders had lower serum myoglobin concentrations (384 ± 243 vs. 597 ± 293 ng·ml, p = 0.04). Although the groups were similar in age, anthropometric characteristics, and training habits, total genotype score was higher in low CK responders than in high CK responders (7.7 ± 1.1 vs. 5.5 ± 1.1 point, p < 0.01). A favorable polygenic profile can contribute to reducing the level of muscle damage developed during endurance exercise.

Coelho, DB, Pimenta, EM, Rosse, IC, Veneroso, C, Pussieldi, GDA, Becker, LK, De Oliveira, EC, Carvalho, MRS, and Silami-Garcia, E. Alpha-actinin-3 R577X polymorphism influences muscle damage and hormonal responses after a soccer game. J Strength Cond Res 33(10): 2655-2664, 2019-The purpose of this study was to evaluate indicators of muscle damage and hormonal responses after soccer matches and its relation to alpha-actinin-3 (ACTN3) gene expression (XX vs. RR/RX), considering that the R allele produces alpha-actinin-3 and provides greater muscle strength and power. Thirty players (10 XX and 20 RR/RX) younger than 16 years were evaluated in this study. Blood samples were collected immediately before, after, 2, and 4 hours after the games to assess muscle damage (creatine kinase [CK] and alpha-actin) and hormonal responses (interleukin-6 [IL-6], cortisol, and testosterone). Postgame CK was higher as compared to the pregame values in both groups and it was also higher in the RR/RX (p < 0.05) than in the XX. The concentrations of alpha-actin and IL-6 were similar for both groups and did not change over time. Testosterone was increased after the game only in the RR/RX group (p < 0.05). Cortisol concentrations in group RR/RX were higher immediately after the game than before the game, and 2 and 4 hours after the game the concentration decreased (p < 0.05). The RR and RX individuals presented higher markers of muscle microtrauma and hormonal stress, probably because they performed more speed and power actions during the game, which is a self-regulated activity. From the different responses presented by RR/RX and XX genotypes, we conclude that the genotypic profile should be taken into account when planning training workloads and recovery of athletes.

BACKGROUND alpha-actinin-4 (Actinin-4 or ACTN4), originally identified as an actin-binding protein associated with the biological function of cancer cells, appears to be highly expressed in numerous human epithelial carcinomas, including breast cancer (BC). In the present study we assessed the role of serum ACTN4 as a biomarker for BC diagnosis, as well as the association between ACTN4 levels and clinicopathological features. MATERIAL AND METHODS ACTN4 expression level was measured with quantitative real-time PCR (qRT-PCR) analysis in serum specimens of 128 BC patients and 96 healthy volunteers. χ² testing was conducted to explore the association of ACTN4 levels with clinicopathologic factors. Moreover, the diagnostic value of ACTN4 was analyzed using receiver operating characteristic (ROC) curves. RESULTS Serum ACTN4 level was obviously upregulated in patients with BC compared with healthy controls (P<0.05). High ACTN4 expression was significantly associated with clinical stage (P=0.000), tumor grade (P=0.004), and lymph node status (P=0.024). However, no association was found between ACTN4 expression and age, tumor size, ER status, PR status, or HER-2 status (all P>0.05). The ROC analysis showed that the area under the curve (AUC) of ACTN4 was 0.887 (95%CI: 0.843-0.931), with sensitivity of 80.5% and specificity of 84.4%, and the cutoff value was 1.050. CONCLUSIONS ACTN4 in serum can serve as a clinical predictor in the diagnosis or prediction of clinical outcomes of patients with BC.

The standard therapeutic strategy recommended for locally advanced pancreatic cancer (LAPC) is typically chemotherapy or chemoradiotherapy (CRT). Although the clinical benefit of chemotherapy alone versus CRT for LAPC has been compared in a number of clinical trials, the optimal therapy for LAPC remains unclear. Moreover, the clinical benefit derived from treatment in each clinical trial is a matter of controversy, and the superiority of one treatment over another has yet to be definitively demonstrated. The poor outcomes seen among patients with LAPC owe largely to the emergence of metastatic disease; therefore, accurately evaluating occult distant metastasis before choosing a therapeutic strategy could be expected to help stratify patients with LAPC into the most appropriate treatment regimen, namely local control or systemic therapy. In 1998, we identified the actinin-4 gene (ACTN4) as an actin-binding protein and showed its molecular mechanisms had clinical implications for cancer metastasis. We also identified ACTN4 gene amplification in pancreatic, ovarian, and salivary gland cancer, and demonstrated its utility as a strong prognostic biomarker for stage I lung adenocarcinoma in patients who had never received chemotherapy. Moreover, we recently reported that ACTN4 gene amplification could be a useful biomarker for predicting the efficacy of CRT for LAPC. In the present review, we summarize current knowledge regarding therapeutic strategies for LAPC and discuss the potential development of personalized medicine using ACTN4 measurement for patients with LAPC., (Copyright © 2018. Published by Elsevier B.V.)

Purpose: Exertional rhabdomyolysis can occur in individuals performing various types of exercise but it is unclear why some individuals develop this condition while others do not. Previous investigations have determined the role of several single nucleotide polymorphisms (SNPs) to explain inter-individual variability of serum creatine kinase (CK) concentrations after exertional muscle damage. However, there has been no research about the interrelationship among these SNPs. The purpose of this investigation was to analyze seven SNPs that are candidates for explaining individual variations of CK response after a marathon competition (ACE = 287bp Ins/Del, ACTN3 = p.R577X, CKMM = NcoI, IGF2 = C13790G, IL6 = 174G>C, MLCK = C37885A, TNFα = 308G>A)., Methods: Using Williams and Folland's model, we determined the total genotype score from the accumulated combination of these seven SNPs for marathoners with a low CK response (n = 36; serum CK <400 U·L-1) vs. marathoners with a high CK response (n = 31; serum CK ≥400 U·L-1)., Results: At the end of the race, low CK responders had lower serum CK (290±65 vs. 733±405 U·L-1; P<0.01) and myoglobin concentrations (443±328 vs. 1009±971 ng·mL-1, P<0.01) than high CK responders. Although the groups were similar in age, anthropometric characteristics, running experience and training habits, total genotype score was higher in low CK responders than in high CK responders (5.2±1.4 vs. 4.4±1.7 point, P = 0.02)., Conclusion: Marathoners with a lower CK response after the race had a more favorable polygenic profile than runners with high serum CK concentrations. This might suggest a significant role of genetic polymorphisms in the levels of exertional muscle damage and rhabdomyolysis. Yet other SNPs, in addition to exercise training, might also play a role in the values of CK after damaging exercise.

Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.

The aim of this study is to investigate the role of anti-alpha-actinin antibodies in patients with new-onset systemic lupus erythematosus (SLE). Thirty-six patients with SLE, 16 of whom had lupus nephritis (LN), and 53 healthy controls were included. The clinical and laboratory parameters of patients were collected from medical records or by questionnaire. Serum anti-alpha-actinin Abs was measured by competitive enzyme linked immunosorbent assay (ELISA). Our results show that the OD value of serum anti-alpha-actinin Abs in SLE patients was significantly lower than that in normal controls (1.212 +/- 0.244 vs. 1.364 +/- 0.202, P = 0.002); seven of 36 SLE patients were seropositive for anti-alpha-actinin Abs, which was significantly higher than in normal controls (19.4 vs. 3.8%, P = 0.028). There were no significant differences of clinical parameters between the anti-alpha-actinin Abs-positive patients and the negative patients. The positive rate of the term urine casts, elevated IgM and IgA in anti-alpha-actinin Abs-positive patients were higher than that in the negative patients. The OD values of serum anti-alpha-actinin Abs negatively correlated with disease activity (R(s) = -0.352, P = 0.035). Anti-alpha-actinin Abs may be a useful marker of the disease activity of SLE; in addition, it may be used as a complementary parameter to differentiate LN from SLE without nephritis.

Alpha-actinins are structural proteins of the Z-line. Human skeletal muscle expresses two alpha-actinin isoforms, alpha-actinin-2 and alpha-actinin-3, encoded by their respective genes ACTN2 and ACTN3. ACTN2 is expressed in all muscle fiber types, while only type II fibers, and particularly the type IIb fibers, express ACTN3. ACTN3 (R577X) polymorphism results in loss of alpha-actinin-3 and has been suggested to influence skeletal muscle function. The X allele is less common in elite sprint and power athletes than in the general population and has been suggested to be detrimental for performance requiring high power. The present study investigated the association of ACTN3 genotype with muscle power during 30-s Wingate cycling in 120 moderately to well-trained men and women and with knee extensor strength and fatigability in a subset of 21 men performing isokinetic exercise. Muscle biopsies were obtained from the vastus lateralis muscle to determine fiber-type composition and ACTN2 and ACTN3 mRNA levels. Peak and mean power and the torque-velocity relationship and fatigability output showed no difference across ACTN3 genotypes. Thus this study suggests that R577X polymorphism in ACTN3 is not associated with differences in power output, fatigability, or force-velocity characteristics in moderately trained individuals. However, repeated exercise bouts prompted an increase in peak torque in RR but not in XX genotypes, suggesting that ACTN3 genotype may modulate responsiveness to training. Our data further suggest that alpha-actinins do not play a significant role in determining muscle fiber-type composition. Finally, we show that ACTN2 expression is affected by the content of alpha-actinin-3, which implies that alpha-actinin-2 may compensate for the lack of alpha-actinin-3 and hence counteract the phenotypic consequences of the deficiency.

Introduction: Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood., Materials and Methods: Methods used are a combination of differential and gradient centrifugation steps. The level of purification was determined by nephelometry, FACS, and PCR., Results: We could show that even the P2Y purinoceptor 12 (P2Y(12)) receptor, which undergoes rapid homologous desensitization, was still functional after the purification procedure. The presence of PINCH (particularly interesting new Cys-His protein) and alpha-parvin, which constitute the IPP (ILK-PINCH-parvin) complex together with the integrin-linked kinase (ILK), has been predicted in platelets by proteomic analysis. We could confirm this observation with our purified platelets. Detection of these proteins is an example of the application of this purification protocol that can be used for the verification of proteins postulated by high-throughput studies., Conclusions: The procedure for obtaining purified platelets described here provides an essential, much-needed tool for the comprehensive investigation of platelet proteins and functions.

Idiopathic nephrotic syndrome is the most common glomerular disorder of childhood. Recurrence of nephrotic syndrome immediately following renal transplantation is rapid, results in a high rate of graft loss, and represents the most severe form of nephrotic syndrome. This review discusses the molecular heterogeneity of pediatric nephrotic syndrome across the spectrum of disease activity. A schema is offered for a molecular approach to pediatric nephrotic syndrome, including immune-mediated and structural/genetic factors., (Copyright 2004 Blackwell Munksgaard)

Background: Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib-IX-V and phosphatidylinositol 3-kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5-trisphosphate (PIP3) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3-kinase (PI 3-KIA)., Methods: Platelets were subjected to 120 dynes cm-2 shear stress in a cone-plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3-KIA performed, or lipids extracted for PIP3 measurements. alpha-Actinin was incubated with phosphatidylinositol 4,5-bisphosphate (PIP2), immunoprecipitated, and used as a substrate for in vitro PI 3-KIA activity., Results: Pathological shear stress induces biphasic PIP3 production. In resting platelets, PI 3-KIA associates with alpha-actinin and PIP2. After exposure to shear stress, alpha-actinin and PIP2 rapidly disassociate from PI 3-KIA. PI 3-KIA then gradually re-associates with PIP2 and alpha-actinin, and this complex becomes linked to GpIb alpha through the cytoskeleton. PIP3 production and the observed changes in the association between alpha-actinin, PIP2, and PI 3-KIA are inhibited when VWF binding to GpIb alpha is blocked. In a cell-free system, alpha-actinin binds PIP2 and when the alpha-actinin-PIP2 complex is added to platelet PI 3-KIA, PIP3 production is stimulated., Conclusions: These results suggest that pathological shear-induced VWF binding to GpIb-IX-V stimulates PIP3 production through the assembly of an alpha-actinin-based complex that colocalizes PI 3-KIA with substrate PIP2.

alpha-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012--37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that alpha-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His(6) tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant alpha-actinin protein containing a Tyr --> Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored alpha-actinin phosphorylation. Purified wild type alpha-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of alpha-actinin that cosedimented with actin filaments. These results establish that alpha-actinin is a direct substrate for FAK and suggest that alpha-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.

Alpha-Actinin and myosin were associated into reorganized actin cable networks and partly formed stress fiber-like structures in platelets during surface activation. Double-label immunofluorescence staining using antibodies against alpha-actinin and platelet myosin heavy chain (MHC) showed that alpha-actinin and myosin were colocalized in the cell center at the early stage of activation and dynamically redistributed with shape change. In the later stage, two proteins were colocalized around the granulomeres. alpha-Actinin was also seen beneath the surface membrane while myosin was not. Occasionally, both proteins were segregated, revealed granular staining in the cell body of flattened platelets and often aligned irregular alternate arrangement in the actin cables. Immunoelectron microscopy (immunogold) employing antibodies against MHC and myosin light chain (MLC) demonstrated that myosin, associated with actin cytoskeleton was precisely filamentous (328 nm in average length, 15 nm in width) and bipolar with a central bare zone, since MLCs were located at both ends. Myosin formed a cluster composed of several filaments with repeating alignment, suggesting each cluster corresponded to the granular staining pattern of immunofluorescence. These observations indicated that the organization of alpha-actinin and myosin in actin cables in activated platelets resembled that in stress fibers in various cultured cells., (Copyright 1998 Academic Press.)

Binding of the alpha-granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified 125I-labeled TSP, we observed a strong interaction of [125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, alpha-actinin. We further demonstrated that [125I]TSP interacts with alpha-actinin in a specific manner and with a high affinity (Kd = 6.6 nM) in a solid-phase binding assay.

Several non-muscle tropomyosins have been reported to lack the ability to polymerize in a head-to-tail manner [Dabrowska, R. et al. (1983) J. Muscle Res. Cell Motil. 1, 83-92; Côté, G.P. (1983) Mol. Cell. Biochem. 57, 127-146]. Unlike rabbit skeletal muscle tropomyosin, these proteins could therefore not protect the F-actin microfilaments neither from disassembly or from cross-linking by the other actin-associating factors. However, we have provided evidence that, in vitro, pig platelet tropomyosin, although shorter in molecular length, exhibits the same properties as the muscle protein: it self-associates and forms a 1:6 complex with platelet filamentous actin under physiological conditions [Prulière et al. (1984) J. Muscle Res. Cell Motil. 6, 126]. In this paper, we examine the effects of several other actin-binding proteins on the microfilaments saturated with this non-muscle tropomyosin. Since contractile proteins often vary with the cell type and may require different conditions for their interactions, we have developed a procedure which allows the parallel purification of actin-binding protein (ABP), vinculin, alpha-actinin, gelsolin as well as actin and tropomyosin from the same batch of cells. Thus, using an homogeneous system, we show by viscometry, sedimentation and densitometry, and by electron microscopy, that pig platelet tropomyosin can protect the structure of the microfilaments from the action of the modulating factors to the same extent as rabbit skeletal muscle alpha-tropomyosin. Our data suggest that interaction of ABP, vinculin or alpha-actinin can occur only with the ends of the filaments when F-actin is saturated with tropomyosin, while cross-linking takes place by interactions with sites localized along the entire length of F-actin in the absence of tropomyosin. Moreover, the presence of tropomyosin on F-actin leads to the total inhibition of gelsolin severing activity, although it did not prevent the binding of gelsolin to the F-actin--tropomyosin complex. This suggests that pig platelet as well as skeletal muscle tropomyosins have the ability to increase the strength of the interaction between actin monomers within the filament. This also suggests that the binding sites of gelsolin along the filaments are not localized in the groove of the F-actin helix.

Recently, the contractile protein alpha-actinin was identified in normal human platelets by its antigenic cross-reaction with a monospecific antibody to purified muscle alpha-actinin. In this study, we extend that preliminary identification of platelet alpha-actinin. Amino acid analysis, one-dimensional peptide maps, and silver stain analysis on polyacrylamide gels demonstrate that human platelet alpha-actinin shows a greater degree of similarity to smooth muscle alpha-actinin than to striated muscle alpha-actinin. There is no evidence to suggest that alpha-actinin is a glycoprotein. In addition, we find that thrombasthenic platelets, which are deficient in glycoproteins IIb and IIIa (GPIIb and GPIIIa) contain normal amounts of alpha-actinin, confirming the recent finding that alpha-actinin and GPIIIa are different proteins in human platelets. We demonstrate that both normal and thrombasthenic platelets also contain vinculin, a 130,000-dalton polypeptide found in many cell types at sites of end-on attachment of microfilaments to the plasma membrane. Thus, the thrombasthenic defect in GPIIb and GPIIIa does not diminish the content of either alpha-actinin or vinculin.

Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.

Isolation of human platelet membranes on polylysine beads and selective solubilization of membrane proteins allowed classification of membrane-associated proteins into integral and peripheral proteins. No major integral protein was found that was not exposed on the surface. Glycoprotein Ic was the only surface-exposed protein that behaved as a peripheral protein. The localization and identification of alpha-actinin was performed with an antibody against porcine skeletal muscle alpha-actinin. Human platelet alpha-actinin had an apparent molecular weight of 100 000 and a pI of 5.7-6.3. It was membrane-associated and behaved as a peripheral protein. Immunoisolation on protein A beads, as well as the 'Western Blot' technique applied to two-dimensional gels, demonstrated that alpha-actinin is not identical to GP III as was previously suggested (Gerrard, J.M., Schollmeyer, J.V., Phillips, D.R. and White, J.G. (1979) Am. J. Pathol. 94, 509-528).

In purified solutions of alpha-actinin from human blood platelets, three polypeptides a, b and c, of approximately 100 kDa, were observed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were identified as alpha-actinin subunits on the basis of their cross-reactivity with antibodies against skeletal muscle alpha-actinin and their interaction with F-actin. After electrophoresis in the absence of sodium dodecyl sulfate, six forms were observed: aa, ab, bb, ac, bc, cc. The similarity between the one-dimensional peptide maps of a and b and the absence of b in the presence of calcium-dependent protease inhibitors indicated that b is probably derived from the a subunit. The c subunit differs from the a subunit. The results provide evidence that there are actually only two platelet alpha-actinin subunits a and c which give rise to three isoforms: two homodimers aa and cc, and one heterodimer ac.

Immunofluorescence microscopy has been used to characterize the morphological transitions that occur as platelets spread on a surface. Antibodies to the microfilament-specific proteins, actin, myosin, tropomyosin, alpha-actinin and filamin as well as antibodies to tubulin were used. Antibody to tubulin reveals the marginal band of microtubules as a bright fluorescent ring, the diameter of which decreases at a time coincident with pseudopod formation. The latter process is dictated by the assembly of microfilament bundles. Although the change in morphology of the platelet was not studied in detail, our data support the idea that microfilament reorganization influences the display of the marginal band of microtubules. A further conclusion is that the platelet in spite of its small diameter is a system suitable for immunofluorescence microscopy, a method which allows the rapid and simultaneous screening of many cells.

Purified alpha-actinin from human platelets was digested with Ca2+-activated protease from muscle. The alpha subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, alpha-actinin is cleaved by the endogenous Ca2+-activated protease.

It has been suggested that a platelet protein that is very similar to muscle alpha-actinin is identical to the membrane glycoprotein IIIa (GPIIIa) of platelets and is responsible for anchoring actin filaments directly into the plasma membrane of platelets. To determine if alpha-actinin and GPIIIa are related in platelets, we analyzed the purified proteins on 5% sodium dodecyl sulfate/polyacrylamide gels. The two proteins differ in mobility in both the unreduced and reduced states, and they stain differently with silver stain. In addition, alpha-actinin is a prominent component of the detergent-insoluble cytoskeletons of platelets, whereas GPIIIa is absent from these structures. By using monospecific antisera to the individual proteins, it was demonstrated that alpha-actinin and GPIIIa are immunologically distinct. We conclude that alpha-actinin and GPIIIa are different proteins in human blood platelets and that it is unlikely that alpha-actinin is an integral membrane protein.

A procedure for the isolation of alpha-actinin from human blood platelets is described. Typical yields were 10-13 mg from 48 g of frozen platelets. The purified platelet alpha-actinin has many physico-chemical properties (molecular weight in native state, molecular weight in denaturing conditions, Stokes radius, ellipticities at 208 and 221 nm) similar to those of muscle alpha-actinins. However, in contrast to muscle alpha-actinins, it is composed of isoforms containing subunits of slightly different molecular weights and its effect on actin gelation is calcium-sensitive. These two characteristics are common to other known non-muscle alpha-actinins.

The structural and functional properties of the aa (2 X 97 kDa) and cc (2 X 94 kDa) isoforms of platelet alpha-actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross-reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F-actin (high-speed sedimentation) and cross-link or gel actin filaments (low-speed sedimentation and viscometry), in a calcium-dependent manner. The study of the interaction with F-actin indicates that the binding of 1 molecule of aa or cc alpha-actinin/9-11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCl2 at 0.1 mM strongly inhibits the binding of aa to F-actin and weakly that of cc, while it inhibits similarly the cross-linking of either aa or cc. The cross-linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of aa at 4, 20, 30 and 37 degrees C, respectively. The bb form (2 X 96 kDa), which is a proteolytic product of aa [Y. Gache et al. (1984) Biochem. Biophys. Res. Commun. 124, 877-881], behaves roughly as aa, but the calcium sensitivity of its binding to F-actin is intermediate between that of aa and cc. These results suggest that the effect of Ca2+ concentration on the binding of platelet alpha-actinin to F-actin may be partly dissociated from the effect on the cross-linking.

Effects of gelsolin on the actomyosin system in platelet have been studied. MgATPase activity of platelet actomyosin is enhanced up to two folds by 200 nM of platelet gelsolin in the presence, but not in the absence of Ca ion. The half maximum enhancement is observed at the concentration of Ca2+ around 10(-5) M. The effect of gelsolin to enhance the ATPase activity of actomyosin is potentiated by tropomyosin, which is a Ca2+-insensitive actomyosin enhancer. The results indicate that gelsolin may control the activity of actomyosin system in platelets.

Comparative analyses of the cytoskeletons of resting and stimulated platelets point out the involvement of a 79 kDa polypeptide in the activation step and its increased incorporation during aggregation. It appears as a doublet and cross-reacts with an antibody to chicken gizzard caldesmon, whereas no 150 kDa immunoreactive form was detected. alpha-Actinin and gelsolin were detected only in the aggregation step.

Blood platelets contain a variety of contractile protein species, including the glycoprotein alpha-actinin, which is found at the Z disc in skeletal muscle cells. In the present study, we have considered the possibility that alpha-actinin might be one of several previously described platelet surface glycoproteins. Purified anti-alpha-actinin antibody was found to react strongly with partially purified platelet glycoprotein III, weakly with platelet glycoprotein IIb, and not at all with platelet glycoproteins Ib and IV. Platelets from three siblings with thrombasthenia, a disorder characterized by severe deficiency of platelet glycoproteins IIb and III, were found also to be equally deficient in alpha-actinin. These findings indicate that alpha-actinin and glycoprotein III are identical and suggest that this protein may be an anchor point for actin on the inside of the membrane. Combined with ultrastructural studies of normal and thrombasthenic platelets, the new findings provide a clearer understanding of contraction in single cells and small aggregates.