The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Medical monitoring during the NASA Artificial Gravity-Bed Rest Pilot Study.

The NASA artificial gravity-bed rest pilot study (AGPS) was designed to investigate the efficacy of daily exposure to a +Gz acceleration gradient for counteracting the physiologic decrements induced by prolonged bed rest. Test subjects were continuously monitored by a physician for signs and symptoms of pre-syncope, motion sickness, and arrhythmias while on the centrifuge. In this article, we have summarized the medical monitoring observations that were made during the AGPS and included an assessment of the relative usefulness of the information provided by the various monitoring tools in making a decision to terminate a centrifuge spin.

Centrifugation protocol for the NASA Artificial Gravity-Bed Rest Pilot Study.

We have implemented a 41-day ground-based study to investigate the effects of daily artificial gravity loading on bed rest deconditioned human subjects. Each subject underwent 21 days of 6 degree head-down bed rest. Treatment subjects received 60 min daily doses of inertial mechanical loading (2.5 G at the feet decreasing to 1 G at the heart) produced by a short radius centrifuge. During rotation, the subject's cardiovascular responses were monitored via ECG, blood pressure and pulse oximetry, and subjective assessment of motion sickness and overall health were periodically requested. The subject's weight distribution at the feet was measured using a force plate, and lower leg muscle activity was monitored via surface electromyography. Control subjects were instrumented but did not receive any centrifugation. This paper provides details on the centrifuge protocol development and efficacy.

Gender differences in cardiovascular tolerance to short radius centrifugation.

In preparing for the NASA Artificial Gravity Pilot Study the planned centrifuge loading protocol was tested in 11 ambulatory subjects (6 men, 5 women). Each was subjected to a single 60 min trial with 2.5G of equivalent gravitational load feet and 1G at the level of the mediastinum. Amongst the men, 5/6 completed the trial successfully with no adverse sequelae. However, amongst the women, 4/5 tests were stopped early because of presyncope. Women are known to have a greater predisposition to syncope following orthostatic stress under normal tilt table conditions, during lower body negative pressure and following space flight. Amongst the factors which may have contributed to their lower tolerance to centrifugation are anthropometric factors, the vasoactive effects of sex hormones, catecholamine levels, ability to augment total peripheral resistance in response to orthostatic stress, and structural differences in cardiac anatomy and physiology. However, determining the true cause of this difference will require further investigation.

Dynamic force spectroscopy of glycoprotein Ib-IX and von Willebrand factor.

The first stage in hemostasis is the binding of the platelet membrane receptor glycoprotein (GP) Ib-IX complex to the A1 domain of von Willebrand factor in the subendothelium. A bleeding disorder associated with this interaction is platelet-type von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Ibalpha subunit of GP Ib-IX. Using optical tweezers and a quadrant photodetector, we investigated the binding of A1 to GOF and loss-of-function mutants of GP Ibalpha with mutations in the region containing the two known naturally occurring mutations. By dynamically measuring unbinding force profiles at loading rates ranging from 200-20,000 pN/s, we found that the bond strengths between A1 and GP Ibalpha GOF mutants (233, 235, 237, and 239) were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined (p < 0.05). In addition, mutants 231 and 232 exhibited significantly lower bond strengths with A1 than the wild-type receptors (p < 0.05). We computed unloaded dissociation rate constant (k(off)(0)) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k(off)(0) value of 5.47 +/- 0.25 s(-1) to be significantly greater than the GOF k(off)(0) values and significantly less than the loss-of-function k(off)(0) values. Our data illustrate the importance of the bond kinetics associated with the VWF/GP Ib-IX interaction in hemostasis and also demonstrate the drastic changes in binding that can occur when only a single amino acid of GP Ibalpha is altered.

Adherence of Staphylococcus aureus fibronectin binding protein A mutants: an investigation using optical tweezers.

Bacterial adhesion to extracellular matrix proteins plays a major role in infections of host tissue and medical devices. In some species of gram-positive cocci, this adhesion is mediated by specific molecules present on the bacterial cell surface. We have used optical tweezers to dynamically measure the adhesive force between an individual Staphylococcus aureus bacterium and a fibronectin-coated surface. A bacterium was optically trapped and brought in contact with a 10-microm diameter polystyrene microsphere coated with fibronectin. The force required to detach the cell from the microsphere was measured by tracking the displacement signals of the trapped cell on a quadrant photodiode throughout the detachment process for a series of S. aureus strains expressing fibronectin-binding proteins with various degrees of mutation. The single-bond rupture forces ranged between 15 and 26 pN depending on the extent of mutation. No binding was observed in the strain with the highest degree of mutation. These results confirm that multiple regions of the S. aureus fibronectin adhesin participate in the binding process and provide further insight into the role of these regions in the adhesive process.

P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface.

von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin alpha v beta 3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a alpha v beta 3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF-P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers.

Ultralarge von Willebrand factor (ULVWF) multimers have been implicated in the pathogenesis of the catastrophic microangiopathic disorder, thrombotic thrombocytopenic purpura. Spontaneous ULVWF binding to platelets has been ascribed to increased avidity due to the greatly increased number of binding sites for platelets (the A1 domain) per molecule. To address the mechanism of enhanced ULVWF binding to platelets, we used optical tweezers to study the unbinding forces from the glycoprotein Ib-IX (GP Ib-IX) complex of plasma VWF, ULVWF, and isolated A1 domain. The unbinding force was defined as the minimum force required to pull ligand-coated beads away from their attachment with GP Ib-IX-expressing cells. Beads coated with plasma VWF did not bind to the cells spontaneously, requiring the modulators ristocetin or botrocetin. The force required to break the ristocetin- and botrocetin-induced plasma VWF-GP Ib-IX bonds occurred in integer multiples of 6.5 pN and 8.8 pN, respectively, depending on the number of bonds formed. In contrast, beads coated with either ULVWF or A1 domain bound the cells in the absence of modulators, with bond strengths in integer multiples of approximately 11.4 pN for both. Thus, in the absence of shear stress, ULVWF multimers form spontaneous high-strength bonds with GP Ib-IX, while plasma VWF requires exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1 domain and greater than those of plasma VWF induced by either modulator. Therefore, we suggest that the conformational state of ULVWF multimers is more critical than their size for interaction with platelets.

Measurement of the binding forces between von Willebrand factor and variants of platelet glycoprotein Ibalpha using optical tweezers.

BACKGROUND AND OBJECTIVE: Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction. STUDY DESIGN/MATERIALS AND METHODS: Canine GP Ibalpha does not normally bind to human vWf, and thus canine-human GP Ibalpha chimeras were constructed by sequentially replacing human GP Ibalpha structural regions with their canine counterparts. Chinese hamster ovary (CHO) cells, which are frequently used to express platelet GP complexes, were transfected with the chimeric proteins. Optical tweezers (lambda = 830 nm) were used to investigate bond strengths between vWf and different GP Ibalpha canine-human chimeras. Since vWf does not bind GP Ibalpha without high shear stress, the compounds botrocetin and ristocetin were used to induce binding between human vWf and the chimeras. RESULTS: All human-canine GP Ibalpha chimeras bound to vWf in the presence of botrocetin. Replacement of the N-terminal flanking sequence and the first leucine-rich repeat resulted in lower GP Ibalpha/vWf bond strengths than the wild-type human GP Ibalpha/vWf bond strength (P < 0.05). Chimeras lacking the second leucine-rich repeat did not adhere to vWf with ristocetin acting as modulator. CONCLUSION: The N-terminal flanking sequence and the first leucine-rich repeat of GP Ibalpha were found to be important but not necessary for GP Ibalpha to adhere to vWf. The second leucine-rich repeat was found to be critical for GP Ibalpha to bind vWf and could potentially be used in the development of a novel recombinant anti-thrombotic drugs.